Hypothalamus-specific polypeptides

ABSTRACT

Peptides and polypeptides found in the hypothalamus region of the mammalian brain are described, particularly hypocretin polypeptides and their uses. Hypocretin polypeptides are biologically active and produce electrical changes in neurons, lower body temperature, and reduce food intake.

REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/1023,220, filed Aug. 2, 1996, which isexplicitly incorporated by reference, as are all references citedherein.

GOVERNMENTAL RIGHTS

[0002] This invention was made with governmental support from the UnitedStates Government, National Institutes of Health, Grants GM32355 andNS33396; the United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

[0003] This invention relates to the identification, isolation,sequencing, use, and expression of hypothalamus-specific proteins andfragments thereof.

BACKGROUND OF THE INVENTION

[0004] The hypothalamus, a phylogenetically ancient region of themammalian brain, is responsible for the integration of the centralnervous system and the endocrine system and is particularly related tothe physiological response to stress. In contrast to laminar corticalstructures such as the cerebellum and hippocampus whose final functionsrely on innervation from the thalamus and brain stem, the hypothalamusis organized as a collection of distinct, autonomously active nucleiwith discrete functions. Ablation and electrical stimulation studies andmedical malfunctions have implicated several of these nuclei as centralregulatory centers for major autonomic and endocrine homeostatic systemsmediating processes such as reproduction, lactation, fluid balance,metabolism, and aspects of behaviors, such as circadian rhythinicity,basic emotions, feeding and drinking, mating activities, and responsesto stress, as well as normal development of the immune system (Shepherd,G. M., Neurobiology, 3rd ed. Oxford University Press, New York, 1994).Distinct hormones and releasing factors have been associated with someof these nuclei but, at best, the organizations and molecular operationsof these structures are only partially understood.

[0005] A substantial portion of a mammal's genetic endowment isdedicated to the function of its central nervous system, as evidenced bythe substantial number of mRNAs selectively expressed in the brain(Sutcliffe, J. G., Ann. Rev. Neurosci. 11:157-198, 1988). Many of thesehave been observed to be selectively associated with distinct neuralsubsets. Existing knowledge of the expression of specific hypothalamichormones and releasing factors suggests that ensembles of mRNAsselectively associated with discrete hypothalamic nuclei may encodeproteins singularly associated with the unique functions of thosenuclei.

SUMMARY OF THE INVENTION

[0006] The present invention provides peptides and polypeptides found inthe hypothalamus region of the mammalian brain. Preferably, the peptidesand polypeptides are enriched in the hypothalamus relative to otherregions of the brain. More preferably the peptides and polypeptides arespecific to the hypothalamus. One embodiment is the rat polypeptidehypocretin also referred to as, H35 protein or clone 35 protein (SEQ IDNO: 1) and polypeptide analogs thereof having at least one conservativeamino acid substitution. Another embodiment is the mouse hypocretinpolypeptide (SEQ ID NO: 2) and polypeptide analogs thereof having atleast one conservative amino acid substitution.

[0007] The present invention also provides polynucleotides encodingpeptides and polypeptides found in the hypothalamus region of the brain.Preferably, the polynucleotides encoding peptides and polypeptides areenriched in the hypothalamus relative to other regions of the brain.More preferably the polynucleotides encoding peptides and polypeptidesare specific to the hypothalamus. One embodiment is a polynucleotidechosen from the group consisting of the polynucleotide of SEQ ID NO: 3,a polynucleotide having at least about 95% of its nucleotide sequenceidentical to the polynucleotide of SEQ ID NO: 3, and polynucleotideshybridizing to the polynucleotide of SEQ ID NO: 3. Another embodiment isa polynucleotide chosen from the group consisting of the polynucleotideof SEQ ID NO: 4, a polynucleotide having at least about 95% of itsnucleotide sequence identical to the polynucleotide of SEQ ID NO: 3, andpolynucleotides hybridizing to the polynucleotide of SEQ ID NO: 4.

[0008] Also provided are vectors for the expression of the novelpolynucleotides operably linked to control sequences capable ofdirecting the production of the novel polypeptides in suitable hostcells.

[0009] In other aspects this invention provides pharmaceuticalcompositions of the polynucleotides, polypeptides and peptides,antibodies to the peptides and polypeptides as well as compositionsthereof. This invention also provides assay methods and kits forpracticing the methods, and methods for using the polynucleotides,peptides and polypeptides for diagnostic and therapeutic purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In the Drawings,

[0011]FIG. 1 shows the results of subtractive screening, enriched forsequences selectively expressed in hypothalamus. Replicate dot blots onwhich the indicated masses of plasmid DNA for clones of neuron-specificenolase (NSE), cyclophilin, proopiomelanocortin (POMC), vasopressin, thevector pT7T3D, protein kinase Cδ (PKCδ) and growth hormone (GH) weremanually spotted and hybridized with cDNA probes made from cRNAtranscribed from the target or subtracted libraries, or an equal mixtureof the cerebellum and hippocampus driver libraries. Comparison of thesignal intensities for the vasopressin dilution series dots at severallevels of autoradiographic exposure suggested a 20-to-30 fold increasein the specific activity of vasopressin cDNA.

[0012]FIG. 2. shows the results of cDNA library Southern blotting withclones representative of the four distribution classes. Theelectrophoretic lanes contain the cerebellum first driver library (D1),the hippocampus second driver library (D2), and the hypothalamus targetlibrary (T) cleaved with HaeIII and hybridized with the inserts fromclone 35 (Panel A), clone 10 (Panel B), clone 86 (Panel C) and clone 19(Panel D).

[0013]FIG. 3 distribution of hypothalamic mRNAs. Northern blots withpoly(A)⁺ RNA isolated from extracts of whole brain, olfactory bulb,cerebral cortex, hippocampus, hypothalamus, thalamus, cerebellum,pituitary, liver, kidney and heart were probed with cDNA inserts fromthe indicated clones. A cyclophilin probe was included in the series asa control for comparable blot loading and RNA integrity. The twohypothalamus samples represent inadvertent mixtures of approximatelyequal parts of hypothalamus and striatum. The expression patterns aregrouped into four classes (A,B,C,D). Only the regions of the blotscontaining the hybridized signal are shown.

[0014]FIG. 4 depicts the expression patterns analyzed by in situhybridization, showing coronal sections of rat brains hybridized withsingle stranded RNA probes corresponding to the inserts of A, clone 35;B, clone 6; C, clone 10; D, clone 20; E, clone 29 and F, clone 21.

[0015]FIG. 5 shows a comparison of rat and mouse cDNA and amino acidsequences corresponding to clone 35 and the amino acid sequence of thepeptide hormone secretin. A. The amino acid sequence is listed on thetop line, the rat nucleotide sequence on the second line and the mousenucleotide sequence is listed on the third line. Differences innucleotide sequences are indicated by asterisks below each differentbase, amino acid differences are indicated by alternatives (rat/mouse)listed above the encoding triplets. Tandem basic amino acids (putativesites for proteolytic maturation) are indicated in bold italics, as isthe serine residue most likely to represent the end of the secretionsignal. B. Alignment of hcrt1 and hcrt2 amino acid sequences with theamino acid sequence of secretin. The first 9 amino acid residues ofsecretin have been repeated to indicate apparent circular permutation.The identities between the hypocretins and members of theglucagon/vasoactive intestinal polypeptidelsecretin family (H. -C.Pehmann, R. Goke, B. Goke, Endocrine Reviews, 16, 390 (1995)) areindicated by asterisks; the hcrt1 and hcrt2 consensus residues appearabove the alignment.

[0016]FIG. 6 shows the cDNA and amino acid sequence of clone 29.

[0017]FIG. 7 is a graphical representation of the results of voltageclamp experiments on isolated in vitro rat hypothalamic cells, in whichapplication of 1 μg hcrt2 produced electrical responses in adult but notimmature neurons.

DETAILED DESCRIPTION OF EMBODIMENTS

[0018] The following definitions are set forth to illustrate and definethe meaning and scope of the various terms used to describe theinvention herein. All patents and other publications mentioned in thisspecification are expressly incorporated by reference herein.

[0019] A. Definitions

[0020] Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. The standardpolypeptide nomenclature (described in J. Biol. Chem., 243:3552-59(1969) and adopted at 37 CFR §1.822(b)(2)) that provides one letter andthree letter codes for amino acid residues is used.

[0021] It should be noted that all amino acid residue sequencesrepresented herein by formulae have a left- to-right orientation in theconventional direction of amino terminus to carboxy terminus. Inaddition, the phrase “amino acid residue” is broadly defined to includemodified and unusual amino acids, such as those listed in 37 CFR1.822(b)(4), and incorporated herein by reference. Furthermore, itshould be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues or a covalent bond to an amino-terminalgroup such as NH₂ or acetyl or to a carboxy-terminal group such as COOH.

[0022] Recombinant DNA molecule: a DNA molecule produced by operativelylinking two DNA segments. Thus, a recombinant DNA molecule is a hybridDNA molecule comprising at least two nucleotide sequences not normallyfound together in nature.

[0023] Receptor: A receptor is a molecule, such as a protein,glycoprotein and the like, that can specifically (non-randomly) bind toanother molecule.

[0024] Antibody: The term antibody in its various grammatical forms isused herein to refer to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antibody combining site or paratope. Exemplary antibodymolecules are intact immunoglobulin molecules, substantially intactimmunoglobulin molecules and portions of an immunoglobulin molecule,including those portions known in the art as Fab, Fab′, and F(ab′)_(z.)

[0025] Antibody Combining Site: An antibody combining site is thatstructural portion of an antibody molecule comprised of a heavy andlight chain variable and hypervariable regions that specifically binds(immunoreacts with) an antigen. The term immunoreact in its variousforms means specific binding between an antigenic determinant-containingmolecule and a molecule containing an antibody combining site such as awhole antibody molecule or a portion thereof.

[0026] Monoclonal Antibody: A monoclonal antibody in its variousgrammatical forms refers to a population of antibody molecules thatcontain only one species of antibody combining site capable ofimmunoreacting with a particular epitope. A monoclonal antibody thustypically displays a single binding affinity for any epitope with whichit immunoreacts. A monoclonal antibody may therefore contain an antibodymolecule having a plurality of antibody combining sites, eachimmunospecific for a different epitope, e.g., a bispecific monoclonalantibody.

[0027] Upstream: In the direction opposite to the direction of DNAtranscription, and therefore going from 5′ to 3′ on the non-codingstrand, or 3′ to 5′ on the mRNA.

[0028] Downstream: Further along a DNA sequence in the direction ofsequence transcription or read out, that is traveling in a 3′- to5′-direction along the non-coding strand of the DNA or 5′- to3′-direction along the RNA transcript.

[0029] Polypeptide: A linear series of amino acid residues connected toone another by peptide bonds between the alpha-amino group and carboxygroup of contiguous amino acid residues.

[0030] Protein: A linear series of more than 50 amino acid residuesconnected one to the other as in a polypeptide.

[0031] Substantially Purified or Isolated: When used in the context ofpolypeptides or proteins, the terms describe those molecules that havebeen separated from components that naturally accompany them. Typically,a monomeric protein is substantially pure when at least about 60% to 75%of a sample exhibits a single polypeptide backbone. Minor variants orchemical modifications typically share the same polypeptide sequence. Asubstantially purified protein will typically comprise over about 85% to90% of a protein sample, more usually about 95%, and preferably will beover about 99% pure.

[0032] Protein or polypeptide purity or homogeneity may be indicated bya number of means well known in the art, such as polyacrylamide gelelectrophoresis of a sample, followed by visualization thereof bystaining. For certain purposes, high resolution is needed and highperformance liquid chromatography (HPLC) or a similar means forpurification utilized.

[0033] Synthetic Peptide: A chemically produced chain of amino acidresidues linked together by peptide bonds that is free of naturallyoccurring proteins and fragments thereof.

[0034] Nucleic acid or polynucleotide sequence: includes, but is notlimited to, eucaryotic mRNA, cDNA, genomic DNA, and synthetic DNA andRNA sequences, comprising the natural nucleoside bases adenine, guanine,cytosine, thymidine, and uracil. The term also encompasses sequenceshaving one or more other bases including, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenine, aziridinylcytosine,pseudoisocytosine, 5-carboxyhydroxylmethyl)uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methyl-inosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methyl-aminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, and 2,6-diaminopurine.

[0035] Coding sequence or open reading frame: a polynucleotide ornucleic acid sequence which is transcribed (in the case of DNA) ortranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a translation startcodon at the 5′ (amino) terminus and a translation stop codon at the 3′(carboxy) terminus. A transcription termination sequence will usually belocated 3′ to the coding sequence.

[0036] Nucleic acid control sequences: translational start and stopcodons, promoter sequences, ribosome binding sites, polyadenylationsignals, transcription termination sequences, upstream regulatorydomains, enhancers, and the like, as are necessary and sufficient forthe transcription and translation of a given coding sequence in adefined host cell. Examples of control sequences suitable for eucaryoticcells are promoters, polyadenylation signals, and enhancers. All ofthese control sequences need not be present in a recombinant vector solong as those necessary and sufficient for the transcription andtranslation of the desired gene are present.

[0037] Operably or operatively linked: the configuration of the codingand control sequences so as to perform the desired function. Thus,control sequences operably linked to a coding sequence are capable ofeffecting the expression of the coding sequence. A coding sequence isoperably linked to or under the control of transcriptional regulatoryregions in a cell when DNA polymerase will bind the promoter sequenceand transcribe the coding sequence into mRNA that can be translated intothe encoded protein. The control sequences need not be contiguous withthe coding sequence, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

[0038] Heterologous and exogenous: as they relate to nucleic acidsequences such as coding sequences and control sequences, denotesequences that are not normally associated with a region of arecombinant construct, and are not normally associated with a particularcell. Thus, a “heterologous” region of a nucleic acid construct is anidentifiable segment of nucleic acid within or attached to anothernucleic acid molecule that is not found in association with the othermolecule in nature. For example, a heterologous region of a constructcould include a coding sequence flanked by sequences not found inassociation with the coding sequence in nature. Another example of aheterologous coding sequence is a construct where the coding sequenceitself is not found in nature (e.g., synthetic sequences having codonsdifferent from the native gene). Similarly, a host cell transformed witha construct which is not normally present in the host cell would beconsidered heterologous for purposes of this invention.

[0039] Expression system: polynucleotide sequences containing a desiredcoding sequence and control sequences in operable linkage, so that cellstransformed with these sequences are capable of producing the encodedproduct. In order to effect transformation, the expression system may beincluded on a discrete vector; however, the relevant polynucleotide mayalso be integrated into the host chromosome.

[0040] Vector: a recombinant polynucleotide comprised of single strand,double strand, circular, or supercoiled DNA or RNA. A typical vector maybe comprised of the following elements operatively linked at appropriatedistances for allowing functional gene expression: replication origin,promoter, enhancer, 5′ mRNA leader sequence, ribosomal binding site,nucleic acid cassette, termination and polyadenylation sites, andselectable marker sequences. One or more of these elements may beomitted in specific applications. The nucleic acid cassette can includea restriction site for insertion of the nucleic acid sequence to beexpressed. In a functional vector the nucleic acid cassette contains thenucleic acid sequence to be expressed including translation initiationand termination sites. An intron optionally may be included in theconstruct, preferably ≧100 bp and placed 5′ to the coding sequence.

[0041] A vector is constructed so that the particular coding sequence islocated in the vector with the appropriate regulatory sequences, thepositioning and orientation of the coding sequence with respect to thecontrol sequences being such that the coding sequence is transcribedunder the “control” of the control sequences. Modification of thesequences encoding the particular protein of interest may be desirableto achieve this end. For example, in some cases it may be necessary tomodify the sequence so that it may be attached to the control sequenceswith the appropriate orientation; or to maintain the reading frame. Thecontrol sequences and other regulatory sequences may be ligated to thecoding sequence prior to insertion into a vector. Alternatively, thecoding sequence can be cloned directly into an expression vector whichalready contains the control sequences and an appropriate restrictionsite which is in reading frame with and under regulatory control of thecontrol sequences.

[0042] Suitable marker sequences for identification and isolation ofcorrectly transfected cells include the thymidine kinase (tk),dihydrofolate reductase (DHFR), and aminoglycoside phosphotransferase(APH) genes. The latter imparts resistance to the aminoglycosideantibiotics, such as kanamycin, neomycin, and geneticin. These, andother marker genes such as those encoding chloramphenicolacetyltransferase (CAT) and β-galactosidase (β-gal), may be incorporatedinto the primary nucleic acid cassette along with the gene expressingthe desired therapeutic protein, or the selection markers may becontained on separate vectors and cotransfected.

[0043] The term “biochemically equivalent variations” means protein ornucleic acid sequences which differ in some respect from the specificsequences disclosed herein, but nonetheless exhibit the same orsubstantially the same functionality. In the case of cDNA, for example,this means that modified sequences which contain other nucleic acidsthan those specifically disclosed are encompassed, provided that thealternate cDNA encodes mRNA which in turn encodes a protein of thisinvention. Such modifications may involve the substitution of only a fewnucleic acids, or many. The modifications may involve substitution ofdegenerate coding sequences or replacement of one coding sequence withanother; introduction of non-natural nucleic acids is included.Preferably, the modified nucleic acid sequence hybridizes to and is atleast 95% complementary to the sequence of interest.

[0044] Similarly, in the case of the proteins and polypeptides of thisinvention, alterations in the amino acid sequence which do not affectfunctionality may be made. Such “biochemically equivalent muteins” mayinvolve replacement of one amino acid with another, use of side chainmodified or non-natural amino acids, and truncation. The skilled artisanwill recognize which sites are most amenable to alteration withoutaffecting the basic function.

[0045] The expression products described herein are proteins andpolypeptides having a defined chemical sequence. However, the precisestructure depends on a number of factors, particularly chemicalmodifications common to proteins. For example, since all proteinscontain ionizable amino and carboxyl groups, the protein may be obtainedin acidic or basic salt form, or in neutral form. The primary amino acidsequence may be derivatized using sugar molecules (glycosylation) or byother chemical derivatizations involving covalent or ionic attachmentwith, for example, lipids, phosphate, acetyl groups and the like, oftenoccurring through association with saccharides. These modifications mayoccur in vitro, or in vivo, the latter being performed by a host cellthrough post-translational processing systems. Such modifications mayincrease or decrease the biological activity of the molecule, and suchchemically modified molecules are also intended to come within the scopeof the invention.

[0046] B. Hypocretin Proteins and Polypeptides

[0047] Hypocretin or clone H35, has been cloned in both rat and mouse.The amino acid residue sequence in these two mammalian species is notidentical but is sufficiently similar to permit generalization regardingfunction, and so that one can identify and isolate the hypocretin genein any mammalian species.

[0048] Variations at both the amino acid and nucleotide sequence levelare described in isolates of hypocretin, and such variations are not tobe construed as limiting. For example, allelic variation within amammalian species can tolerate a several percent difference betweenisolates of a type of hypocretin, which differences comprisenon-deleterious variant amino acid residues. Thus a protein of about 95%homology, and preferably at least 98% homology, to a disclosedhypocretin is considered to be an allelic variant of the disclosedhypocretin, and therefore is considered to be a hypocretin of thisinvention.

[0049] As disclosed herein, hypocretin is produced first in vivo inprecursor form, and is then processed into smaller polypeptides havingbiological activity as described herein. Insofar as these differentpolypeptide forms are useful, the term hypocretin protein or polypeptideconnotes all species of polypeptide having an amino acid residuesequence derived from the hypocretin gene.

[0050] The complete coding nucleotide sequence, clone 35, of rat H35cDNA is 569 nucleotides in length, and is listed in SEQ ID NO 3. Thecomplete preprohypocretin cDNA clone presents a 390 nucleotide openreading frame (ORF) plus triplet termination codons (FIG. 5). There is aN-terminal signal peptide with a cleavage site between amino acidpositions 27 and 28, corresponding to a cleavage site after nucleotideposition 172 of SEQ ID NO: 3.

[0051] Translation of this rat cDNA sequence produces a novel protein of130 amino acid residues, referred to as rat preprohypocretin. The aminoacid sequence of rat preprohypocretin is listed in SEQ ID NO: 1. Theamino acid sequence of mouse preprohypocretin is listed in SEQ ID NO: 2.

[0052] A hypocretin protein of this invention can be in a variety offorms, depending upon the use therefor, as described herein. Forexample, a hypocretin an be isolated from a natural tissue.

[0053] Alternatively, a hypocretin protein of this invention can be arecombinant protein, that is, produced by recombinant DNA methods asdescribed herein. A recombinant hypocretin protein need not necessarilybe substantially pure, or even isolated, to be useful in certainembodiments, although recombinant production methods are a preferredmeans to produce a source for further purification to yield an isolatedor substantially pure receptor composition. A recombinant hypocretinprotein can be present in or on a mammalian cell line or in crudeextracts of a mammalian cell line.

[0054] In one embodiment, a hypocretin protein is substantially free ofother neuropeptides, so that the purity of a hypocretin reagent, andthus freedom from pharmacologically distinct proteins, facilitates usein the screening methods. The recombinant production methods are ideallysuited to produce significantly improved purity in this regard, althoughbiochemical purification methods from natural sources are also included.In this regard, a hypocretin protein is substantially free from otherneuropeptides if there are insufficient other neuropeptides such thatpharmacological cross-reactivity is not detected in conventionalscreening assays for ligand binding or biological activity.Alternatively, recombinant hypocretin fusion proteins can be produced byjoining nucleotides encoding additional amino acid residue sequence inproper reading frame at the 3′ end of the hypocretin sequence. Thefusion protein thus produced exhibits properties of the added amino acidsequence in addition to the properties of hypocretin. For example, theadditional amino acid sequence may serve to help identify and purity therecombinantly produced hypocretin fusion protein. One preferredhypocretin fusion protein is hypocretin-poly(His).

[0055] Preferably, a hypocretin protein of this invention is present ina composition in an isolated form, i.e., comprising at least about 0.1percent by weight of the total composition, preferably at least 1%, andmore preferably at least about 90%. Particularly preferred is asubstantially pure preparation of hypocretin, that is at least 90% byweight, and more preferably at least 99% by weight. Biochemical methodsuseful for the enrichment and preparation of an isolated hypocretinbased on the chemical properties of a polypeptide are well known, andcan be routinely used for the production of proteins which are enrichedby greater than 99% by weight.

[0056] An isolated or recombinant hypocretin protein of this inventioncan be used for a variety of purposes, as described further herein. Ahypocretin protein can be used as an immunogen to produce antibodiesimmunoreactive with hypocretin. Hypocretin proteins can be used in invitro ligand binding assays for identifying ligand bindingspecificities, and agonists or antagonists thereto, to characterizecandidate pharmaceutical compounds useful for modulating hypocretinfunction, and as therapeutic agents for effecting hypocretin functions.Other uses will be readily apparent to one skilled in the art.

[0057] Furthermore, the invention includes analogs of a hypocretinprotein of this invention. An analog is a man-made variant whichexhibits the qualities of a hypocretin of this invention in terms ofimmunological reactivity, ligand binding capacity or the like functionalproperties of a hypocretin protein of this invention. An analog cantherefore be a cleavage product of hypocretin, can be a polypeptidecorresponding to a portion of hypocretin, can be hypocretin polypeptidein which a membrane anchor has been removed, and can be a varianthypocretin sequence in which some amino acid residues have been altered,to name a few alternatives.

[0058] Insofar as the present disclosure identifies hypocretin fromdifferent mammalian species, the present invention is not to be limitedto a hypocretin protein derived from one or a few mammalian species.Thus, the invention includes a mammalian hypocretin protein, which canbe derived, by recombinant DNA or biochemical purification from naturalsources, from any of a variety of species including man, mouse, rabbit,rat, dog, cat, sheep, cow, and the like mammalian species, withoutlimitation. Human and agriculturally relevant animal species areparticularly preferred.

[0059] Exemplary hypocretin species identified herein are rat and mousehypocretin.

[0060] The amino acid reside sequence of rat preprohypocretin is shownin SEQ ID NO 1, and corresponding nucleotide (cDNA) of ratpreprohypocretin is shown in SEQ ID NO 3.

[0061] The amino acid residue sequence of mouse preprohypocretin isshown in SEQ ID NO 2, and corresponding nucleotide (cDNA) of mousepreprohypocretin is shown in SEQ ID NO 4.

[0062] A hypocretin protein of this invention can be prepared by avariety of means, although expression in a mammalian cell using arecombinant DNA expression vector is preferred. Exemplary productionmethods for a recombinant hypocretin are described in the Examples.

[0063] The invention also provides a method for the production ofisolated hypocretin proteins, either as intact hypocretin protein, asfusion proteins or as smaller polypeptide fragments of hypocretin. Theproduction method generally involves inducing cells to express ahypocretin protein of this invention, recovering the hypocretin from theresulting cells, and purifying the hypocretin so recovered bybiochemical fractionation methods, using a specific antibody of thisinvention, or other chemical procedures.

[0064] The inducing step can comprise inserting a recombinant DNA vectorencoding a hypocretin protein, or fragment thereof, of this invention,which recombinant DNA is capable of expressing a hypocretin, into asuitable host cell, and expressing the vector's hypocretin gene.

[0065] As used herein, the phrase “hypocretin polypeptide” refers to apolypeptide having an amino acid residue sequence that comprises anamino acid residue sequence that corresponds, and preferably isidentical, to a portion of a hypocretin of His invention.

[0066] A hypocretin polypeptide of this invention is characterized byits ability to immunologically mimic an epitope (antigenic determinant)expressed by a hypocretin of this invention. Such a polypeptide isuseful herein as a component in an inoculum for producing antibodiesthat immunoreact with native hypocretin and as an antigen in immunologicmethods. Representative and preferred hypocretin polypeptides for use asan inununogen in an inoculum are shown herein.

[0067] As used herein, the phrase “immunologically mimic” in its variousgrammatical forms refers to the ability of a hypocretin polypeptide ofthis invention to immunoreact with an antibody of the present inventionthat recognizes a conserved native epitope of a hypocretin as definedherein.

[0068] It should be understood that a subject polypeptide need not beidentical to the amino acid residue sequence of a hypocretin receptor,so long as it includes the required sequence.

[0069] In addition, certain hypocretin polypeptides derived fromreceptor binding portions of hypocretin have the capacity to inhibit thebinding of the hypocretin that would normally bind a hypocretinreceptor. Thus, the invention also includes hypocretin polypeptideswhich are specifically designed for their capacity to mimic exposedregions of hypocretin involved in hypocretin receptor bindinginteractions and thereby receptor function. Therefore, thesepolypeptides have the capacity to function as analogs to hypocretin, andthereby block function.

[0070] In addition, polypeptides corresponding to exposed domains havethe ability to induce antibody molecules that immunoreact with ahypocretin of this invention at portions of hypocretin involved inreceptor protein function, and therefor the antibodies are also usefulat modulating normal hypocretin function.

[0071] A hypocretin polypeptide is preferably no more than about 120amino acid residues in length for reasons of ease of synthesis. Thus, itmore preferred that a hypocretin polypeptide be no more that about 100amino acid residues, still more preferably no more than about 50residues, and optimally less than 40 amino acid residues in length whensynthetic methods of production are used. Exemplary polypeptides arehcrt1 and hcrt2.

[0072] The present invention also includes a hypocretin polypeptide thathas an amino acid residue sequence that corresponds to the sequence ofthe hypocretin protein shown in the sequence listings, and includes anamino acid residue sequence represented by a formula selected from thegroup consisting of the polypeptides shown in the sequence listings. Inthis embodiment, the polypeptide is further characterized as having theability to mimic a hypocretin epitope and thereby inhibits hypocretinfunction in a classic hypocretin receptor activation assay, as describedherein.

[0073] Due to the three dimensional structure of a native foldedhypocretin molecule, the present invention includes that multipleregions of hypocretin are involved in hypocretin receptor function,which multiple and various regions are defined by the various hypocretinpolypeptides described above, A preferred hypocretin receptor ligand ishcrt. The ability of the above-described polypeptides to inhibitreceptor-ligand binding can readily be measured in a ligand bindingassay as is shown in the Examples herein. Similarly, the ability of theabove-described polypeptides to inhibit hypocretin receptor function canreadily be measured in a receptor assay as is described herein.

[0074] In another embodiment, the invention includes hypocretinpolypeptide compositions that comprise one or more of the differenthypocretin polypeptides described above which inhibit hypocretinreceptor function, admixed in combinations to provide simultaneousinhibition of multiple contact sites on the hypocretin receptor.

[0075] A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is capable of mimicking an epitope ofhypocretin. Therefore, a present polypeptide can be subject to variouschanges, substitutions, insertions, and deletions where such changesprovide for certain advantages in its use. In this regard, a hypocretinpolypeptide of this invention corresponds to, rather than is identicalto, the sequence of a hypocretin protein where one or more changes aremade and it retains the ability to induce antibodies that immunoreactwith a hypocretin of this invention.

[0076] The term “analog” includes any polypeptide having an amino acidresidue sequence substantially identical to a sequence specificallyshown herein in which one or more residues have been conservativelysubstituted with a functionally similar residue and which displays theability to induce antibody production as described herein. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another, the substitution of one polar (hydrophilic) residue foranother such as between arginine and lysine, between glutamine andasparagine, between glycine and serine, the substitution of one basicresidue such as lysine, arginine or histidine for another, or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another.

[0077] The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite binding activity.

[0078] “Chemical derivative” refers to a subject polypeptide having oneor more residues chemically derivatized by reaction of a functional sidegroup. Such derivatized molecules include for example, those moleculesin which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For examples: 4hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.D-amino acids may also be included in place of one or more L-aminoacids. Polypeptides of the present invention also include anypolypeptide having one or more additions and deletions or residuesrelative to the sequence of a polypeptide whose sequence is shownherein, so long as the requisite activity is maintained.

[0079] The term “fragment” refers to any subject polypeptide having anamino acid residue sequence shorter than that of a polypeptide whoseamino acid residue sequence is shown herein.

[0080] When a polypeptide of the present invention has a sequence thatis not identical to the sequence of a hypocretin polypeptide, it istypically because one or more conservative or non-conservativesubstitutions have been made, usually no more than about 30 numberpercent, more usually no more than 20 number percent, and preferably nomore than 10 number percent of the amino acid residues are substituted.Additional residues may also be added at either terminus for the purposeof providing a “linker” by which the polypeptides of this invention canbe conveniently affixed to a label or solid matrix, or carrier.Preferably the linker residues do not form a hypocretin epitope, i.e.,are not similar is structure to a hypocretin protein.

[0081] Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described hereinbelow.

[0082] Amino acid residue linkers are usually at least one residue andcan be 40 or more residues, more often 1 to 10 residues, but do not forma hypocretin epitope. Typical amino acid residues used for linking aretyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. Inaddition, a subject polypeptide can differ, unless otherwise specified,from the natural sequence of a hypocretin protein by the sequence beingmodified by terminal-NH₂ acylation, e.g., acetylation, or thioglycolicacid amidation, by terminal-carboxlyamidation, e.g., with ammonia,methylamine, and the like.

[0083] When coupled to a carrier to form what is known in the art as acarrier-hapten conjugate, a hypocretin polypeptide of the presentinvention is capable of inducing antibodies that immunoreact withhypocretin. In view of the well established principle of immunologiccross-reactivity, the present invention therefore includes antigenicallyrelated variants of the polypeptides shown herein. An “antigenicallyrelated variant” is a subject polypeptide that is capable of inducingantibody molecules that immunoreact with a polypeptide described hereinand with a hypocretin protein of this invention.

[0084] Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

[0085] Suitable bases capable of forming salts with the peptides of thepresent invention include inorganic bases such as sodium hydroxide,ammonium hydroxide, potassium hydroxide and the like; and organic basessuch as mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

[0086] A hypocretin polypeptide of the present invention, also referredto herein as a subject polypeptide, can be synthesized by any of thetechniques that are known to those skilled in the polypeptide art,including recombinant DNA techniques. Synthetic chemistry techniques,such as a solid-phase Merrifield-type synthesis, are preferred forreasons of purity, antigenic specificity, freedom from undesired sideproducts, case of production and the like. An excellent summary of themany techniques available can be found in J. M. Steward and J. D. Young,“Solid Phase Peptide Synthesis”, W. H. Freeman Co., San Francisco, 1969;M. Bodansky, et al., “Peptide Synthesis”, John Wiley & Sons, SecondEdition, 1976 and J. Meienhofer, “Hornonal Proteins and Peptides”, Vol.2, p. 46, Academic Press (New York), 1983 for solid phase peptidesynthesis, and E. Schroder and K. Kubke, “The Peptides”, Vol. 1,Academic Press (New York), 1965 for classical solution synthesis, eachof which is incorporated herein by reference. Additional peptidesynthesis methods are described by Sutcliffe in U.S. Pat. Nos. 4,900,811and 5,242,798, which are hereby incorporated by reference. Appropriateprotective groups usable in such synthesis are described in the abovetexts and in J. F. W. McOmie, “Protective Groups in Organic Chemistry”,Plenum Press, New York, 1973, which is incorporated herein by reference.

[0087] In general, the solid-phase synthesis methods comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

[0088] Using a solid phase synthesis as exemplary, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the finalpolypeptide.

[0089] A hypocretin polypeptide can be used, inter alia, in thediagnostic methods and systems of the present invention to detect ahypocretin receptor or hypocretin itself present in a body sample, orcan be used to prepare an inoculum as described herein for thepreparation of antibodies that immunoreact with conserved epitopes onhypocretin.

[0090] In addition, certain of the hypocretin polypeptides of thisinvention can be used in the therapeutic methods of the presentinvention to inhibit hypocretin function as described further herein.

[0091] C. Nucleic Acids and Polynucleotides

[0092] The DNA segments of the present invention are characterized asincluding a DNA sequence that encodes a hypocretin protein of thisinvention. That is, the DNA segments of the present invention arecharacterized by the presence of some or all of a hypocretin structuralgene. Preferably the gene is present as an uninterrupted linear seriesof codons where each codon codes for an amino acid residue found in thehypocretin protein, i.e., a gene free of introns.

[0093] One preferred embodiment is a DNA segment that codes an aminoacid residue sequence that defines a hypocretin protein as definedherein, and the DNA segment is capable of expressing a hypocretinprotein of this invention. A preferred DNA segment codes for an aminoacid residue sequence substantially the same as, and preferablyconsisting essentially of, an amino acid residue sequence shown in thesequence listing for a hypocretin protein, such as in SEQ ID NOs 1 and2.

[0094] The amino acid residue sequence of a protein or polypeptide isdirectly related via the genetic code to the deoxyribonucleic acid (DNA)sequence of the structural gene that codes for the protein. Thus, astructural gene or DNA segment can be defined in terms of the amino acidresidue sequence, i.e., protein or polypeptide, for which it codes.

[0095] An important and well known feature of the genetic code is itsdegeneracy. That is, for most of the amino acids used to make proteins,more than one coding nucleotide triplet (codon) can code for ordesignate a particular amino acid residue. Therefore, a number ofdifferent nucleotide sequences may code for a particular amino acidresidue sequence. Such nucleotide sequences are considered functionallyequivalent since they can result in the production of the same aminoacid residue sequence in all organisms. Occasionally, a methylatedvariant of a purine or pyrimidine may be incorporated into a givennucleotide sequence. However, such methylations do not affect the codingrelationship in any way.

[0096] A nucleic acid is any polynucleotide or nucleic acid fragment,whether it be a polyribonucleotide of polydeoxyribonucleotide, i.e., RNAor DNA, or analogs thereof. In preferred embodiments, a nucleic acidmolecule is in the form of a segment of duplex DNA, i.e, a DNA segment,although for certain molecular biological methodologies, single-strandedDNA or RNA is preferred.

[0097] DNA segments (i.e., synthetic oligonucleotides) that encodeportions of hypocretin proteins can easily be synthesized by chemicaltechniques, for example, the phosphotriester method of Matteucci, etal., (J. Am. Chem. Soc., 103:3185-3191, 1981) or using automatedsynthesis methods. In addition, larger DNA segments can readily beprepared by well known methods, such as synthesis of a group ofoligonucleotides that define the DNA segment, followed by hybridizationand ligation of oligonucleotides to build the complete segment.

[0098] Of course, by chemically synthesizing the coding sequence, anydesired modifications can be made simply by substituting the appropriatebases for those encoding the native amino acid residue sequence.

[0099] Furthermore, DNA segments consisting essentially of structuralgenes encoding a hypocretin protein can be obtained from recombinant DNAmolecules containing a gene that defines a hypocretin protein of thisinvention, and can be subsequently modified, as by site directedmutagenesis, to introduce any desired substitutions.

[0100] 1. Cloning Hypocretin Genes

[0101] Hypocretin genes of this invention can be cloned by a variety ofcloning methods and from any mammalian species. The cloning is based onthe observation that there is a significant degree of homology betweenmammalian species for any given hypocretin of this invention, andtherefor can be conducted according to the general methods described inthe Examples, using nucleic acid homology strategies.

[0102] A typical degree of homology required to successfully clone ahypocretin is at least about 80% homologous at the DNA level, and atleast about 90% homologous at the protein level. Preferred cloningstrategies for isolating a nucleic acid molecule that encodes ahypocretin molecule of this invention are described in the Examples, andincludes the recitation of polynucleotide probes useful for thescreening of libraries of nucleic acid molecules believed to contain atarget hypocretin gene.

[0103] Sources of libraries for cloning a hypocretin gene of thisinvention can include genomic DNA or messenger RNA (mRNA) in the form ofa cDNA library from a tissue believed to express a hypocretin of thisinvention. Preferred tissues are brain tissues, particularlyhypothalamic tissue. The similarities between rat and mouse hypocretinare further extended to the identification of a sequence of iteration oftrinucleotide CTG repeats. For both mammals, a sequence of fouriterations of the trinucleotide CTG repeats followed by two pairs of CTGare present encoding leucine residues. Thus, the presence of theiterations is typically located within the coding region for the signalpeptide.

[0104] Such a triplet expansion in other genes has been implicated ascausal in neurological diseases, e.g., myotonic dystrophy as describedby Brook et al., Cell, 68:799-808 (1992) and fragile-X syndrome asdescribed by Fu et al., Cell, 67:1047-1058 (1991). In myotonic dystrophypatients who are mildly affected, at least 50 CTG repeats are present.In severely affected individuals, the expansion can exist up to severalkilobase pairs. In contrast, in the normal population, the repeatsequence is highly variable ranging from 5 to 27 copies.

[0105] Individuals with varying severities of fragile-X have beensimilarly characterized.

[0106] Screening for the presence of a region of DNA in which therepeats are present in either normal, underexpansion or overexpansionform can provide a genetic basis for diagnosis for some diseases. Thesame may be true for hypocretin in that expansion of the region maycontribute to the basis for a neuronal disorder or disease of the brainor other tissue.

[0107] 2. Oligonucleotides

[0108] The invention also includes oligonucleotides useful for methodsto detect the presence of a hypocretin gene or gene transcript (mRNA) ina tissue by diagnostic detection methods based on the specificity ofnucleic acid hybridization or primer extension reactions. One embodimentincludes any polynucleotide probe having a sequence of a portion of ahypocretin gene of this invention, or a related and specific sequence.Hybridization probes can be of a variety of lengths from about 10 to5000 nucleotides long, although they will typically be about 20 to 500nucleotides in length. Hybridization methods are extremely well known inthe art and will not be described further here.

[0109] In a related embodiment, detection of hypocretin genes can beconducted by primer extension reactions such as the polymerase chainreaction (PCR). To that end, PCR primers are utilized in pairs, as iswell known, based on the nucleotide sequence of the gene to be detected.Particularly preferred PCR primers can be derived from any portion of ahypocretin DNA sequence, but are preferentially from regions which arenot conserved in other cellular proteins.

[0110] A preferred PCR primer pair useful for detecting hypocretin genesand hypocretin gene expression are described in the Examples. Nucleotideprimers from the corresponding region of hypocretin described herein arereadily prepared and used as PCR primers for detection of the presenceor expression of the corresponding gene in any of a variety of tissues.

[0111] 3. Expression Vectors

[0112] In addition, the invention includes a recombinant DNA molecule(recombinant DNA) containing a DNA segment of this invention encoding ahypocretin protein as described herein. A recombinant DNA can beproduced by operatively linking a vector to a DNA segment of the presentinvention.

[0113] The choice of vector to which a DNA segment of the presentinvention is operatively linked depends directly, as is well known inthe art, on the functional properties desired, e.g., protein expression,and the host cell to be. transformed, these being limitations inherentin the art of constructing recombinant DNA molecules. However, a vectorof the present invention is at least capable of directing thereplication, and preferably also expression, of a hypocretin structuralgene included in DNA segments to which it is operatively linked.

[0114] In one embodiment, a vector of the present invention includes aprocaryotic replicon, i.e., a DNA sequence having the ability to directautonomous replication and maintenance of the recombinant DNA moleculeextrachromosomally in a procaryotic host cell, such as a bacterial hostcell, transformed therewith. Such replicons are well known in the art.In addition, those embodiments that include a procaryotic replicon alsoinclude a gene whose expression confers drug resistance to a bacterialhost transformed therewith. Typical bacterial drug resistance genes arethose that confer resistance to ampicillin or tetracycline.

[0115] Those vectors that include a procaryotic replicon can alsoinclude a procaryotic promoter capable of directing the expression(transcription and translation) of a hypocretin gene in a bacterial hostcell, such as E. coli, transformed therewith. A promoter is anexpression control element formed by a DNA sequence that permits bindingof RNA polymerase and transcription to occur. Promoter sequencescompatible with bacterial hosts are typically provided in plasmidvectors containing convenient restriction sites for insertion of a DNAsegment of the present invention. Typical of such vector plasmids arepUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories,(Richmond, Calif.), pRSET available from Invitrogen (San Diego, Calif.)and pPL and pKK223 available from Pharmacia, Piscataway, N.J.

[0116] Expression vectors compatible with eucaryotic cells, preferablythose compatible with vertebrate cells, can also be used to form therecombinant DNA molecules of the present invention. Eucaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia),pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC,#31255), pRc/CMV (Invitrogen, Inc.), the vector pCMV4 described herein,and the like eucaryotic expression vectors.

[0117] In preferred embodiments, the eucaryotic cell expression vectorsused to construct the recombinant DNA molecules of the present inventioninclude a selection marker that is effective in an eucaryotic cell,preferably a drug resistance selection marker. A preferred drugresistance marker is the gene whose expression results in neomycinresistance, i.e., the neomycin phosphotransferase (neo) gene. Southernet al., J. Mol. Appl. Genet., 1:327-341 (1982). Alternatively, theselectable marker can be present on a separate plasmid, and the twovectors are introduced by co-transfection of the host cell, and selectedby culturing in the appropriate drug for the selectable marker.

[0118] 4. Inhibitory Nucleic Acids

[0119] In accordance with one embodiment of the invention, nucleic acidmolecules can be used in methodologies for the inhibition of hypocretingene expression, thereby inhibiting the function of thehypocretin:hypocretin receptor binding interaction by blockinghypocretin expression.

[0120] To that end, the invention includes isolated nucleic acidmolecules, preferably single-stranded nucleic acid molecules(oligonucleotides), having a sequence complementary to a portion of astructural gene encoding a hypocretin protein of this invention. Nucleicacid-based inhibition is well known and generally referred to as“anti-sense” technology by virtue of the use of nucleotide sequenceshaving complementarily which can hybridize to the “sense” strand ormRNA, and thereby perturb gene expression. Typical oligonucleotides forthis purpose are about 10 to 5,000, preferably about 20-1000,nucleotides in length and have a sequence capable of hybridizingspecifically with a structural protein region of the nucleotide sequencethat encodes a hypocretin protein of this invention.

[0121] In one embodiment, the invention includes repetitive units of thenucleotide sequence complementary to a portion of a hypocretinstructural gene so as to present multiple sites for complementarybinding to the structural gene. This feature may be provided in a singlenucleic acid segment having repeating sequences defining multipleportions of a structural gene, by physical conjugation of DNA segmentseach containing a single portion of a structural gene, or a combinationthereof comprising conjugates of DNA segments, each having one or moresequences complementary to a structural gene.

[0122] Nucleotide base modifications can be made to provide certainadvantages to a DNA segments of this invention, referred to asnucleotide analogs. A nucleotide analog refers to moieties whichfunction similarly to nucleotide sequences in a nucleic acid molecule ofthis invention but which have non-naturally occurring portions. Thus,nucleotide analogs can have altered sugar moieties or inter-sugarlinkages. Exemplary are the phosphorothioate and other sulfur-containingspecies, analogs having altered base units, or other modificationsconsistent with the spirit of this invention.

[0123] Preferred modifications include, but are not limited to, theethyl or methyl phosphorate modifications disclosed in U.S. Pat. No.4,469,863 and the phosphorothioate modified deoxyribonucleotidesdescribed by LaPlanche et al., Nucl. Acids Res., 14:9081, 1986; and Stecet al., J. Am. Chem. Soc., 106:6077, 1984. These modifications provideresistance to nucleolytic degradation, thereby contributing to theincreased half-life in therapeutic modalities. Preferred modificationsare the modifications of the 3′-terminus using phosphothioate (PS)sulfurization modification described by Stein et al., Nucl. Acids Res.,16:3209, 1988.

[0124] In accordance with the methods of this invention in certainpreferred embodiments, at least some of the phosphodiester bonds of thenucleotide sequence can be substituted with a structure which functionsto enhance the ability of the compositions to penetrate into the regionof cells where the hypocretin structural gene to be inhibited islocated. It is preferred that such linkages be sulfur containing asdiscussed above, such as phosphorotioate bonds. Other substitutions caninclude alkyl phosphothioate bonds, N-alkyl phosphoramidates,phosphorodithioates, alkyl phosphonates, and short chain alkyl orcycloalkyl structures. In accordance with other preferred embodiments,the phosphodiester bonds are substituted with structures which are, atonce, substantially non-ionic and non-chiral.

[0125] D. Anti-Hypocretin Antibodies

[0126] An antibody of the present invention, i.e., an anti-hypocretinantibody, in one embodiment is characterized as comprising antibodymolecules that immunoreact with a hypocretin protein of this invention.Preferably, an antibody further immunoreacts with a hypocretin proteinin situ, i.e., in a tissue section.

[0127] The invention describes an anti-hypocretin antibody thatimmunoreacts with any of the hypocretin polypeptides of this invention,preferably also immunoreacts with the corresponding recombinanthypocretin protein, and more preferably also reacts with a nativeprotein in situ in a tissue section. Preferably, the antibody issubstantially free from immunoreaction with other proteins orneuropeptides other than hypocretin. Assays for immunoreaction usefulfor assessing immunoreactivity are described herein.

[0128] In one embodiment, antibody molecules are described thatimmunoreact with a hypocretin receptor polypeptide of the presentinvention and that have the capacity to immunoreact with an exposed siteon hypocretin that is required for hypocretin receptor binding. Thus,preferred antibody molecules in this embodiment also inhibit hypocretinreceptor function, and are therefore useful therapeutically to block thereceptor's function.

[0129] Exemplary hypocretin inhibitory antibodies immunoreact with ahypocretin polypeptide described herein that defines an exposed regionof a hypocretin protein that is involved in hypocretin receptorfunction, such as ligand binding.

[0130] An antibody of the present invention is typically produced byimmunizing a mammal with an inoculum containing a hypocretin polypeptideof this invention and thereby induce in the mammal antibody moleculeshaving immunospecificity for the immunizing polypeptide. The antibodymolecules are then collected from the mammal and isolated to the extentdesired by well known techniques such as, for example, by using DEAESephadex to obtain the IgG fraction. Exemplary antibody preparationmethods using hypocretin polypeptides in the immunogen are describedherein in the Examples.

[0131] The preparation of antibodies against polypeptide is well knownin the art. See Staudt et al., J. Exp. Med., 157:687-704 (1983), or theteachings of Sutcliffe, J. G., as described in U.S. Pat. No. 4,900,811,the teachings of which are hereby incorporated by reference.

[0132] Briefly, to produce a hypocretin peptide antibody composition ofthis invention, a laboratory mammal is inoculated with animmunologically effective amount of a hypocretin polypeptide, typicallyas present in a vaccine of the present invention. The anti-hypocretinantibody molecules thereby induced are then collected from the mammal asan antiserum and those immunospecific for both a hypocretin polypeptideand the corresponding recombinant hypocretin protein are isolated to theextent desired by well known techniques such as, for example, byimmunoaffinity chromatography. Alternatively, the antiserum may be used.

[0133] To enhance the specificity of the antibody, the antibodies arepreferably purified by immunoaffinity chromatography using solidphase-affixed immunizing polypeptide. The antibody is contacted with thesolid phase-affixed immunizing polypeptide for a period of timesufficient for the polypeptide to immunoreact with the antibodymolecules to form a solid phase-affixed immunocomplex. The boundantibodies are separated from the complex by standard techniques.

[0134] The word “inoculum” in its various grammatical forms is usedherein to describe a composition containing a hypocretin polypeptide ofthis invention as an active ingredient used for the preparation ofantibodies against a hypocretin polypeptide. When a polypeptide is usedin an inoculum to induce antibodies it is to be understood that thepolypeptide can be used in various embodiments, e.g., alone or linked toa carrier as a conjugate, or as a polypeptide polymer. However, for easeof expression and in context of a polypeptide inoculum, the variousembodiments of the polypeptides of this invention are collectivelyreferred to herein by the term “polypeptide” and its various grammaticalforms.

[0135] For a polypeptide that contains fewer than about 35 amino acidresidues, it is preferable to use the peptide bound to a carrier for thepurpose of inducing the production of antibodies.

[0136] One or more additional amino acid residues can be added to theamino- or carboxy-termini of the polypeptide to assist in binding thepolypeptide to a carrier. Cysteine residues added at the amino- orcarboxy-terrini of the polypeptide have been found to be particularlyuseful for forming conjugates via disulfide bonds. However, othermethods well known in the art for preparing conjugates can also be used.

[0137] The techniques of polypeptide conjugation or coupling throughactivated functional groups presently known in the art are particularlyapplicable.

[0138] See, for example, Aurameas, et al., Scand. J. Immunol., Vol. 8,Suppl. 7:7-23 (1978) and U.S. Pat. No. 4,493,795, U.S. Pat. No.3,791,932 and U.S. Pat. No. 3,839,153. In addition, a site-directedcoupling reaction can be carried out so that any loss of activity due topolypeptide orientation after coupling can be minimized. See, forexample, Rodwell et al., Biotech., 3:889-894 (1985), and U.S. Pat. No.4,671,958.

[0139] Exemplary additional linking procedures include the use ofMichael addition reaction products, di-aldehydes such as glutaraldehyde,Klipstein, et al., J. Infect. Dis., 147:318-326 (1983) and the like, orthe use of carbodiinide technology as in the use of a water-solublecarbodiimide to form amide links to the carrier. Alternatively, theheterobifunctional cross-linker SPDP(N-succinimidyl-3-(2-pyridyldithio)proprionate)) can be used toconjugate peptides, in which a carboxy-terminal cysteine has beenintroduced.

[0140] Useful carriers are well known in the art, and are generallyproteins themselves. Exemplary of such carriers are keyhole limpethemocyanin (KLH), edestin, thyroglobulin, albumins such as bovine serumalbumin (BSA) or human serum albumin (HSA), red blood cells such assheep erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well aspolyamino acids such as poly D-lysine:D-glutamic acid, and the like.

[0141] The choice of carrier is more dependent upon the ultimate use ofthe inoculum and is based upon criteria not particularly involved in thepresent invention. For example, a carrier that does not generate anuntoward reaction in the particular animal to be inoculated should beselected.

[0142] The present inoculum contains an effective, immunogenic amount ofa polypeptide of this invention, typically as a conjugate linked to acarrier. The effective amount of polypeptide per unit dose sufficient toinduce an immune response to the immunizing polypeptide depends, amongother things, on the species of animal inoculated, the body weight ofthe animal and the chosen inoculation regimen is well known in the art.Inocula typically contain polypeptide concentrations of about 10micrograms (μg) to about 500 milligrams (mg) per inoculation (dose),preferably about 50 micrograms to about 50 milligrams per dose.

[0143] The term “unit dose” as it pertains to the inocula refers tophysically discrete units suitable as unitary dosages for animals, eachunit containing a predetermined quantity of active material calculatedto produce the desired immunogenic effect in association with therequired diluent; i.e., carrier, or vehicle. The specifications for thenovel unit dose of an inoculum of this invention are dictated by and aredirectly dependent on (a) the unique characteristics of the activematerial and the particular immunologic effect to be achieved, and (b)the limitations inherent in the art of compounding such active materialfor immunologic use in animals, as disclosed in detail herein, thesebeing features of the present invention.

[0144] Inocula are typically prepared from the dried solidpolypeptide-conjugate by dispersing the polypeptide-conjugate in aphysiologically tolerable (acceptable) diluent such as water, saline orphosphate-buffered saline to form an aqueous composition.

[0145] Inocula can also include an adjuvant as part of the diluent.Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund'sadjuvant (IFA) and alum are materials well known in the art, and areavailable commercially from several sources.

[0146] The antibody so produced can be used, inter alia, in thediagnostic methods and systems of the present invention to detecthypocretin present in a sample such as a tissue section or body fluidsample. Anti-hypocretin antibodies that inhibit hypocretin function canalso be used in vivo in therapeutic methods as described herein.

[0147] A preferred anti-hypocretin antibody is a monoclonal antibody. Apreferred monoclonal antibody of this invention comprises antibodymolecules that immunoreact with a hypocretin polypeptide of the presentinvention as described for the anti-hypocretin antibodies of thisinvention. More preferably, the monoclonal antibody also immunoreactswith recombinantly produced whole hypocretin protein.

[0148] A monoclonal antibody is typically composed of antibodiesproduced by clones of a single cell called a hybridoma- that secretes(produces) only one kind of antibody molecule. The hybridoma cell isformed by fusing an antibody-producing cell and a myeloma or otherself-perpetuating cell line. The preparation of such antibodies wasfirst described by Kohler and Milstein, Nature, 256:495-497 (1975), thedescription of which is incorporated by reference. The hybridomasupernates so prepared can be screened for the presence of antibodymolecules that immunoreact with a hypocretin polypeptide, or forinhibition of hypocretin binding to hypocretin receptor as describedherein.

[0149] Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a hypocretin antigen, such as is present in ahypocretin polypeptide of this invention. The polypeptide-inducedhybridoma technology is described by Niman et al., Proc. Natl. Acad.Sci., USA, 80:4949-4953 (1983), the description of which is incorporatedherein by reference.

[0150] It is preferred that the myeloma cell line used to prepare ahybridoma be from the same species as the lymphocytes. Typically, amouse of the strain 129 GIX⁺ is the preferred mammal. Suitable mousemyelomas for use in the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American TypeCulture Collection, Rockville, Md., under the designations CRL 1580 andCRL 1581, respectively.

[0151] Splenocytes are typically fused with myeloma cells usingpolyethylene glycol (PEG) 1500. Fused hybrids are selected by theirsensitivity to HAT. Hybridomas producing a monoclonal antibody of thisinvention are identified using the enzyme linked immunosorbent assay(ELISA) described in the Examples.

[0152] A monoclonal antibody of the present invention can also beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that produces and secretesantibody molecules of the appropriate polypeptide specificity. Theculture is maintained under conditions and for a time period sufficientfor the hybridoma to secrete the antibody molecules into the medium. Theantibody-containing medium is then collected. The antibody molecules canthen be further isolated by well known techniques.

[0153] Media useful for the preparation of these compositions are bothwell known in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's Minimal Essential Medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mM glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

[0154] Other methods of producing a monoclonal antibody, a hybridomacell, or a hybridoma cell culture are also well known. See, for example,the method of isolating monoclonal antibodies from an immunologicalrepertoire as described by Sastry, et al., Proc. Natl. Acad. Sci. USA,86:5728-5732 (1989); and Huse et al., Science, 246:1275-1281 (1989).

[0155] The monoclonal antibodies of this invention can be used in thesame manner as disclosed herein for antibodies of the present invention.

[0156] For example, the monoclonal antibody can be used in thetherapeutic, diagnostic or in vitro methods disclosed herein whereimmunoreaction with hypocretin is desired.

[0157] Also included in this invention is the hybridoma cell, andcultures containing a hybridoma cell that produce a monoclonal antibodyof this invention.

[0158] E. Diagnostic Methods

[0159] The present invention includes various assay methods fordetermining the presence, and preferably amount, of hypocretin in a bodysample such as a tissue sample, including tissue mass or tissue section,or in a biological fluid sample using a polypeptide, polyclonal antibodyor monoclonal antibody of this invention as an immunochemical reagent toform an immunoreaction product whose amount relates, either directly orindirectly, to the amount of hypocretin in the sample.

[0160] Those skilled in the art will understand that there are numerouswell known clinical diagnostic chemistry procedures in which animmunochemical reagent of this invention can be used to form animmunoreaction product whose amount relates to the amount of hypocretinin a body sample. Thus, while exemplary assay methods are describedherein, the invention is not so limited.

[0161] For example, in view of the demonstrated property that hypocretinbinds a hypocretin receptor, a hypocretin protein of this invention canbe used directly as a probe for detection of a hypocretin receptor bybinding thereto.

[0162] Additionally, one can use a nucleic acid molecule probesdescribed herein to detect the presence in a cell or tissue of ahypocretin gene or expressed gene in the form of mRNA encoding ahypocretin protein of this invention, as described further herein.Suitable probe-based assays are described by Sutcliffe in U.S. Pat. Nos.4,900,811 and 5,242,798, the disclosures of which are incorporated byreference.

[0163] Various heterogenous and homogeneous protocols, eithercompetitive or noncompetitive, can be employed in performing an assaymethod of this invention.

[0164] For example, one embodiment includes a method for assaying theamount of hypocretin protein in a sample that utilizes ananti-hypocretin antibody to immunoreact with hypocretin protein in asample. In this embodiment, the antibody immunoreacts with hypocretin toform a hypocretin-antibody immunoreaction complex, and the complex isdetected indicating the presence of hypocretin in the sample.

[0165] An immunoassay method using an anti-hypocretin antibody moleculefor assaying the amount of hypocretin in a sample typically comprisesthe steps of:

[0166] (a) Forming an immunoreaction admixture by admixing (contacting)a sample with an anti-hypocretin antibody of the present invention,preferably a monoclonal antibody. The sample is typically in the form ofa fixed tissue section in a solid phase such that the immunoreactionadmixture has both a liquid phase and a solid phase, and the antibodyfunctions as a detection reagent for the presence of hypocretin in thesample.

[0167] Preferably, the sample is a brain tissue sample that has beenprepared for immunohistological staining as is well known, althoughother tissue samples may be adsorbed onto a solid phase, includingtissue extracts or body fluid. In that case the adsorption onto a solidphase can be conducted as described for well known Western blotprocedures.

[0168] (b) The immunoreaction admixture is maintained under biologicalassay conditions for a predetermined time period such as about 10minutes to about 16-20 hours at a temperature of about 4 degree Celsiusto about 45 degree Celsius that, such time being sufficient for thehypocretin present in the sample to immunoreact with (immunologicallybind) the antibody and form a hypocretin-containing immunoreactionproduct (immunocomplex).

[0169] Biological assay conditions are those that maintain thebiological activity of the immunochemical reagents of this invention andthe hypocretin sought to be assayed. Those conditions include atemperature range of about 4 degree Celsius to about 45 degree Celsius,a pH value range of about 5 to about 9 and an ionic strength varyingfrom that of distilled water to that of about one molar sodium chloride.Methods for optimizing such conditions are well known in the art.

[0170] (c) The presence, and preferably amount, of hypocretin-containingimmunoreaction product that formed in step (b) is determined (detected),thereby determining the amount of hypocretin present in the sample.

[0171] Determining the presence or amount of the immunoreaction product,either directly or indirectly, can be accomplished by assay techniqueswell known in the art, and typically depend on the type of indicatingmeans used.

[0172] Preferably, the determining of step (c) comprises the steps of:

[0173] (i) admixing the hypocretin-containing immunoreaction productwith a second antibody to form a second (detecting) immunoreactionadmixture, said second antibody molecule having the capacity toimmunoreact with the first antibody (primary) in the immunoreactionproduct.

[0174] Antibodies useful as the second antibody include polyclonal ormonoclonal antibody preparations raised against the primary antibody.

[0175] (ii) maintaining said second immunoreaction admixture for a timeperiod sufficient for said second antibody to complex with theimmunoreaction product and form a second immunoreaction product, and

[0176] (iii) determining the amount of second antibody present in thesecond immunoreaction product and thereby the amount of immunoreactionproduct formed in step (c).

[0177] In one embodiment, the second antibody is a labeled antibody(i.e., detecting antibody) such that the label provides an indicatingmeans to detect the presence of the second immunoreaction productformed. The label is measured in the second immunoreaction product,thereby indicating the presence, and preferably amount, of secondantibody in the solid phase.

[0178] Alternatively, the amount of second antibody can be determined bypreparation of an additional reaction admixture having an indicatingmeans that specifically reacts with (binds to) the second antibody, asis well known. Exemplary are third immunoreaction admixtures with alabeled anti-immunoglobulin antibody molecule specific for the secondantibody. After third immunoreaction, the formed third immunoreactionproduct is detected through the presence of the label.

[0179] Exemplary methods involve the use of in situ immunoreactionmethods using tissue sections, or Western blot procedures, as describedby Sutcliffe in U.S. Pat. No. 4,900,811.

[0180] Another embodiment is a method for assaying the amount oftherapeutically administered hypocretin protein or anti-hypocretinantibody in a body fluid sample such as cerebrospinal fluid (CSF),blood, plasma or serum. The method utilizes a competition reaction inwhich either a hypocretin polypeptide or an anti-hypocretin antibodymolecule of this invention is present in the solid phase as animmobilized immunochemical reagent, and the other of the two reagents ispresent in solution in the liquid phase, in the form of a labeledreagent. A fluid sample is admixed thereto to form a competitionimmunoreaction admixture, and the resulting amount of label in the solidphase is proportional, either directly or indirectly, to the amount ofhypocretin polypeptide or antibody in the fluid sample, depending uponthe format.

[0181] One version of this embodiment comprises the steps of:

[0182] (a) Forming a competition immunoreaction admixture by admixing(contacting) a fluid sample with:

[0183] (1) an anti-hypocretin antibody according to this inventioncontaining antibody molecules that immunoreact with a hypocretin proteinof this invention, said antibody being operatively linked to a solidmatrix such that the competition immunoreaction admixture has both aliquid phase and a solid phase, and

[0184] (2) a polypeptide or recombinant hypocretin protein of thepresent invention that is immunoreactive with the added antibody. Theadmixed polypeptidelprotein in the liquid phase (labeled competingantigen) is operatively linked to an indicating means as describedherein.

[0185] (b) The competition immunoreaction admixture is then maintainedfor a time period sufficient for the competing antigen and the bodysample antigen present in the liquid phase to compete for immunoreactionwith the solid phase antibody. Such immunoreaction conditions arepreviously described, and result in the formation of an indicatingmeans-containing immunoreaction product comprising the labeled competingantigen in the solid phase.

[0186] (c) The amount of indicating means present in the product formedin step (b) is then determined, thereby determining the presence, andpreferably amount, of sample antigen present in the fluid sample.

[0187] Determining the indicating means in the solid phase is thenconducted by the standard methods described herein.

[0188] A reverse version of this embodiment comprises the steps of:

[0189] (a) Forming a competition immunoreaction admixture by admixing afluid sample with:

[0190] (1) an anti-hypocretin antibody according to the presentinvention; and

[0191] (2) a hypocretin polypeptide or recombinant hypocretin protein ofthe present invention (capture antigen) that is immunoreactive with theantibody and is operatively linked to a solid matrix such that thecompetition immunoreaction admixture has both a liquid phase and a solidphase.

[0192] (b) The competition immunoreaction admixture is then maintainedfor a time period sufficient for any hypocretin antigen oranti-hypocretin antibody in the fluid to compete with the admixedantibody molecules for immunoreaction with the solid phase captureantigen and form an antibody-containing immunoreaction product in thesolid phase.

[0193] (c) The amount of antibody present in the product formed in step(b) is then determined, thereby determing the presence and amount oftarget material in the fluid sample.

[0194] In preferred embodiments, the antibody is operatively linked toan indicating means such that the determining in step (c) comprisesdetermining the amount of indicating means present in the product formedin step (b).

[0195] Preferably, the fluid sample is provided to a competitionimmunoreaction admixture as a known amount of CSF, blood, or a bloodderived product such as serum or plasma. Further preferred areembodiments wherein the amount of immunochemical reagent in the liquidphase of the immunoreaction admixture is an excess amount relative tothe amount of reagent in the solid phase. Typically, a parallel set ofcompetition immunoreactions are established using a known amount ofpurified recombinant hypocretin or polypeptide in a dilution series sothat a standard curve can be developed, as is well known. Thus, theamount of product formed in step (c) when using a fluid sample iscompared to the standard curve, thereby determining the amount of targetantigen present in the fluid.

[0196] In another embodiment, the method for assaying the amount ofhypocretin in a sample utilizes a first capture antibody to capture andimmobilize hypocretin in the solid phase and a second indicator antibodyto indicate the presence of the captured hypocretin antigen. In thisembodiment, one antibody immunoreacts with a hypocretin protein to forma hypocretin-antibody immunoreaction complex, and the other antibody isable to immunoreact with the hypocretin while present in thehypocretin-antibody immunoreaction complex. This embodiment can bepracticed in two formats with the immobilized capture antibody beingeither of the two above-identified antibodies, and the indicatorantibody being the other of the two antibodies.

[0197] Where an antibody is in the solid phase as a capture reagent, apreferred means for determining the amount of solid phase reactionproduct is by the use of a labeled hypocretin polypeptide, followed bythe detection means described herein for other labeled products in thesolid phase.

[0198] Also included are immunological assays capable of detecting thepresence of immunoreaction product formation without the use of a label.Such methods employ a “detection means”, which means are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelpolypeptides, methods and systems. Exemplary detection means includemethods known as biosensors and include biosensing methods based ondetecting changes in the reflectivity of a surface, changes in theabsorption of an evanescent wave by optical fibers or changes in thepropagation of surface acoustical waves.

[0199] Alternative methods of expression, amplification, andpurification will be apparent to the skilled artisan. Representativemethods are disclosed in Sambrook, Fritsch, and Maniatis, eds. MolecularCloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory(1989) and in Ausabel et al., eds., Current Protocols in MolecularBiology, Wiley & Sons, Inc., New York (1989).

[0200] D. Specific Methods

[0201] Directional tag PCR subtractive hybridization was used to enricha cDNA library for clones of mRNA species selectively expressed in thehypothalamus. Candidate clones identified by their hybridization to asubtracted hypothalamus probe were validated in three stages. First, ahigh throughput cDNA library Southern blot was used to demonstrate thatthe candidate corresponded to a species enriched in the subtractedlibrary. Second, candidate clones positive in the first assay were usedas probes for Northern blots with RNA from several brain regions andperipheral tissues. Finally, candidate clones that were still positivewere subjected to in situ hybridization analysis to detect thehypothalamic regions that express the corresponding mRNAs.

[0202] Typically, subtractive hybridization protocols utilize a singletarget-driver dichotomy for enrichment of target-specific species. Inthe current study, a two-step subtraction protocol, first depletinghypothalamus sequences with a cerebellum driver, and then with ahippocampus driver, was employed. Previous studies using single stepsubtraction methodology had been successful in finding clones of speciesenriched in a target compared to the single driver tissue, only to findconsiderable expression in other brain regions. The present protocol wasdesigned to provide a more stringent selection for clones of mRNAs withhigh selectivity for the target. Grids of the subtracted library wereprepared and probed as described by Usui, H., Falk, J. D., Dopazo, A.,de Lecea, L., Erlander, M. G., & Sutcliffe, J. G., J. Neurosci.14:4915-4926 (1994). DNA sequence analysis, Northern blotting and insitu hybridization were performed as described by Usui et al., supra,and de Lecea, L., Soriano, E., Criado, J. R., Steffensen, S. C.,Henriksen, S. J., & Sutcliffe, J. G., Molec. Brain Res 25:286-296(1994).

[0203] In situ hybridization analysis was performed essentially asdescribed by Gall, C. M. & Isackson, P. M., Science 245: 758-761 (1989)and by Erlander, M. G., et al., Proc. Natl. Acad. Sci. USA 90: 3452-3456(1993). Coronal sections about 25 μm thick cut from brains of adultSprague-Dawley rats were hybridized at 55 degrees Celsius for 16 hourswith ³⁵S-labelled single-stranded RNA probes at 10⁷ counts per minuteper ml. Free-floating sections were treated with Rnase A at 4 μg/ml at37 degrees Celsius for 1 hour and washed in 1×SSC (15 mM NaCl, 1.5 mM Nacitrate), 50% formamide at 55 degrees Celsius for 2 hours. Finalstringency washes were in 0.1×SSC at 68 degrees Celsius for 1 hour.Sections were mounted on coated slides, dehydrated and exposed to KodakXAR film for 5 days at room temperature.

[0204] For cDNA library Southern blotting, 2 μg of each library wasdigested with HaeIII, separated by electrophoresis, transferred to nylonmembranes, and hybridized to individual clones, as described in Usui etal, supra.

[0205] To recognize mRNAs that are selectively expressed in thehypothalamus, poly(A)-enriched cytoplasmic RNA from carefully dissectedrat and mouse hypothalami were prepared. Target cDNA libraries in vectorpT7T3D (Pharmacia Biotech, Piscataway, N.J.) and driver libraries inpGEM11Zf(−) (Promega, Madison, Wis.) from analogously preparedcerebellar and hippocampal RNA samples were constructed. The directionaltag PCR subtractive hybridization method of Usui and colleagues in Usuiet al., supra was applied to produce tagged hypothalamic cDNAs fromwhich cerebellar and hippocampal sequences were depleted in twoconsecutive steps, removing more than 97% of the input target cDNA. Thetag sequences were used as PCR primer-binding sites to amplify theremaining material. An aliquot of the amplified product was cloned intopBCSK⁺ (Stratagene, La Jolla, Calif.) to generate a subtractedhypothalamus library with 5×10⁵ members, with inserts ranging from 400to 1200 (average 700) nucleotide pairs, as judged by agarose gelelectrophoresis of the released inserts.

[0206] To validate the efficiency of the subtraction, the degree ofdepletion in the subtracted library of sequences known to be expressedpanneurally and the enrichment of sequences known to be expressedspecifically in the hypothalamus was determined. Dot blots were preparedwith dilutions of cDNA clones of the mRNAs encoding the followingproteins: panneural neuron-specific enolase, ubiquitously expressedcyclophilin, hypothalamus-specific vasopressin, hypothalamus-enrichedproopiomelanocortin (POMC), thalamus-specific protein kinase Cδ, andpituitary-specific growth hormone, as well as the target vector itself.The blots were probed with cDNA inserts amplified by PCR from theunsubtracted target library, the subtracted target library or a pool ofthe driver libraries (FIG. 1). The driver and unsubtracted-libraryprobes gave strong signals for cyclophilin and neuron-specific enolase,and a weaker signal for POMC. Neither hippocampus nor cerebellum isknown to express POMC. Although this finding could be explained if oneof the drivers had suffered contamination with mRNA from anotherstructure, for example brain stem, the studies below suggest that thesignal with the driver libraries was probably due to backgroundhybridization to sequences in the POMC clone. The unsubtracted targetadditionally gave a weak signal for vasopressin. The subtracted probegave a very strong signal for vasopressin and POMC and otherwise onlyfaint or undetectable signals. The increase in strength of thevasopressin signal was 20-to-30 fold. Thus, the subtraction protocolremoved abundant, panneurally expressed sequences nearly quantitativelywhile enriching for hypothalamus-specific sequences. There was noapparent contamination with sequences from the anatomically adjacentstructures, thalamus or pituitary. The effectiveness of the subtractionwas quantitated further by measuring the frequencies of VAT-1 andoxytocin clones in the unsubtracted and subtracted libraries by colonyhybridization with a probe corresponding to a mixture of clones of thesetwo species. The frequency of positive clones in the unsubtracted targetwas 4/2775. After subtraction, the frequency increased to 33/1224. Thesefrequencies indicate an approximately 19-fold increase in the specificactivities of these known hypothalamus-enriched species, consistent withthe estimates suggested by the data of FIG. 1.

[0207] To identify species enriched by the subtraction, 648 clones fromthe subtracted library were placed into grid arrays and hybridized tothree replicate blots of grid images with probes prepared from theunsubtracted or subtracted target library, or a pool of the driverlibraries. Approximately 70% of the colonies gave significant signalswith the subtracted target probe compared to 50% with the unsubtractedtarget probe. Only 10% of the colonies gave signals with themixed-driver probe.

[0208] Plasmid DNA was prepared individually from 100 of the coloniesthat gave strong signals with target-derived probes but no signal withthe mixed-driver probe. Partial sequences of the inserts were determinedfor 94 of these, using a sequencing primer that annealed to the vectorregion adjacent to the 3′ ends of the inserts. The remaining 6 cloneswere not pursued further because clear sequences were not obtained. Morethan 90% of the 3′ sequences appeared to be derived from bona fide 3′ends of mRNAs as they contained recognizable poly(A)-addition consensushexads (Birnstiel, M. L., Busslinger, M., & Strub, K. Cell 41:349-359,1985) 12-22 nucleotides upstream from the poly(A) tracts used in theirdirectional cloning. The sequences were searched by BLAST analysis(Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D., J.Molec. Biol. 215:403, 1990) against the GenBank database. For those thatappeared to be novel, the sequence at the 5′ end of the insert was alsodetermined and compared with the database.

[0209] A compilation of those data is presented in Table 1 and databaseaccession numbers are given for those prototypes for which a match wasfound. The 94 clones from the subtracted library for which data wereobtained corresponded to 43 distinct mRNA species. Twenty-nine of thesewere encountered only once in the set of 94 clones, while 14 specieswere seen between 2 to 13 times. Among the 43 distinct species were 21that were unambiguously matched to known mRNA species and 22 that werenovel species. Amongst the novel species were 6 that appear tocorrespond (greater than 80% nucleotide sequence identity across anextensive span) to rat homologues of so-called “expressed sequence tags”(ESTs), mRNAs of as yet unknown function compiled in the databases. Twospecies exhibited similarities in both their partial nucleotidesequences and putative encoded amino acid sequences that suggest them torepresent members of protein families: a protein related to the VAT-1secretory vesicle protein (clone 6), and a new calmodulin-dependentprotein kinase (clone 29, SEQ ID NO: 5).

[0210] The cDNA insert from at least one representative of each of the43 mRNA species was used as a probe in a Southern blot with lanescorresponding to the hypothalamus, hippocampus and cerebellum target anddriver cDNA libraries, each cleaved with the restriction endonucleaseHaeIII. Assuming that the cDNA libraries are representative of the mRNAsexpressed in their corresponding tissues, this assay serves as a lowcost, high throughput surrogate for more expensive and time consumingNorthern blot analyses. The hybridization results of the clones in thisso-called “cDNA library Southern blot” assay were classified in one offive patterns (Table 1): hybridization to bands detected exclusively inthe hypothalamus library (A), to bands highly enriched in hypothalamusbut still detectable in hippocampus and cerebellum lanes (B), to bandsin hypothalamus and hippocampus but not in cerebellum (C), to bands in.all three tissues (D), or too faint to categorize (E). Examples ofclasses A-D are shown in FIG. 2. Twenty-three of the 43 distinct mRNAspecies were exclusive to or highly enriched in the hypothalamuslibrary, and an additional 15 species were undetectable in thecerebellum library, indicating the effectiveness of the protocols foridentifying species selectively present in the target library. It may besignificant that the patterns classified as D corresponded to clonesthat were isolated only once; similarly, none of the species lacking apoly(A)-addition signal turned up more than once. The existence in thecollection of species present in hippocampal, but not cerebellar,libraries presumably is explained by their enrichment during the firstsubtraction step with cerebellum driver to an extent that did not allowtheir complete depletion in the second step with hippocampus driver.POMC gave an A pattern in this assay, demonstrating that the driverlibraries were not significantly contaminated with POMC-expressingstructures. Thus the low POMC signal observed with the driver probes inFIG. 1 is mostly likely accounted for by vector cross-hybridization.

[0211] Northern blots were performed for 15 of the species that showedhypothalamus-enriched or -specific distributions (group A or B) in thecDNA Southern blot assay. The blots (FIG. 3) included RNA samples from 6grossly dissected regions of rat brain in addition to pituitary, liver,kidney and heart. For the clones of species that had been isolated twoor more times, the correspondence with the cDNA library Southern blotassay was excellent. Thus, clones 2 (oxytocin) and 35 (novel), whichgave A patterns in the cDNA Southern blot study, each detected a bandthat was strong in the hypothalamus lanes, but only very faint orundetectable in the other lanes. The faint signals were possibly due tolow expression in those tissues or to contamination during tissuedissection. Clones 6 (VAT1-like), 10 (novel) and 12 (novel), which hadgiven B patterns, each detected bands that were considerably moreintense in the hypothalamus than hippocampus or cerebellum lanes,although each was detected in the pituitary lane (6 strongly) and in thesamples from some other structures. Clones 3 (novel), 15 (novel) and 29(novel calmodulin-dependent protein kinase), although classifiedoriginally as 13 patterns, are more properly considered as C patterns,as their expression profiles in this assay are not enriched inhypothalamus per se, but rather are low in the cerebellum.

[0212] The clones encountered only once behaved, as a group, less well.Clones 21 (novel), 37 (novel), 98 (novel) and 99 (kinesin) failed toshow substantial enrichment in hypothalamus over hippocampus orcerebellum (although 98 was thalamus enriched). However, clone 33(novel) detected an RNA species more prevalent in hypothalamus andthalamus than cortex, pons or olfactory bulb and was undetectable inhippocampus, cerebellum or peripheral tissues; thus, technicallyspeaking, clone 33 maintained its A pattern classification. Clone 20(novel) detected an RNA species with ubiquitous expression butenrichment in hypothalamus and thalamus, thus it is more properlyclassified as B pattern. Clone 67 (novel) detected a species enriched inhypothalamus and olfactory bulb that was detectable in other brainregions and pituitary but was not detectable in cerebellum.

[0213] In situ hybridization on coronal sections of brain from adultmale rats was performed using the inserts from clones representing allfour classes (A-D): 6, 10, 20, 21, 29 and 35. For all clones, thehybridization pattern was consistent with the Northern blot data. In theA class, the clone 35 mRNA displayed a striking pattern of bilaterallysymmetric expression restricted to a few cells in the paraventricularhypothalamic area and ependymal cells surrounding the brain ventricles.No clone 35 signals were detected outside the hypothalamus. The sequenceof clone 35 is shown in FIG. 5.

[0214] Clones 6, 10 and 20, belonging to class B. displayed somewhatmore complex distributions. Clone 6 gave strong signals in theperiventricular hypothalamic nucleus, anterior hypothalamic area,preoptic and arcuate nuclei. Very strong hybridization could also beseen in the centromedial thalamic nucleus and medial habenula. Clone 10displayed almost the same pattern but additional strong signals could beseen in the laterodorsal thalamic nucleus and dentate gyrus, with weaksignals in the hippocampal CA fields and the entire neocortex.Interestingly this mRNA showed a marked enrichment in basal diencephalicstructures that included nuclei not only of the hypothalamus but also ofthe amygdaloid complex. Clone 20 exhibited low levels of expression inseveral areas of the brain, but displayed especially strong signals inthe ventral hypothalamus, most notably in the anterior hypothalamic andperiventricular nuclei.

[0215] Clone 29 (class C, SEQ ID NO: 5), which encodes a novelcalmodulin kinase-like protein, was also very strongly expressed in theanterior hypothalamic area and arcuate nucleus, as well as in thepyramidal cell layer of all hippocampal fields and in the medial andcentral nuclei of the amygdala. The sequence of clone 29 is shown inFIG. 6. Clone 21 represents a class D cDNA, whose distribution includeshypothalamic as well as extrahypothalamic structures. In particular, theclone 21 mRNA was found in cortex, amygdala, hippocampus, caudate, andseveral thalamic (centrodorsal and reticular nuclei) and hypothalamicnuclei. Within the hypothalamus, clone 21 mRNA was especially abundantin the paraventricular hypothalamic nucleus.

[0216] The data compiled in Table 1 suggest that this strategy waseffective: 53 of the 94 clones studied were shown to correspond to mRNAsexpressed in the hypothalamus at much higher concentrations than ineither the hippocampus or cerebellum. An additional 32 of the cloneswere enriched in both hypothalamus and hippocampus over cerebellum,indicating that the first subtraction was more efficient, probablybecause the target concentration was higher in the hybridizationreaction, thus a greater portion of the common species were driven intohybrids. Cumulatively, 85 of the 94 candidates were found to be enrichedin the target hypothalamus compared to the cerebellum, a quiteacceptable success rate. It is noteworthy that in 8 cases, the cDNAlibrary Southern blot assay suggested a higher degree of hypothalamusenrichment than was later observed by Northern blotting, presumably dueto artifactual enrichment in the target libraries compared to the driverlibraries. In a few cases this can be explained by artifactual cloningof an internal or intronic cDNA fragment. Other cases may be explainedby difficulties in achieving proportional representation of lowprevalence mRNAs in cDNA libraries.

[0217] The subtraction steps provided an approximately 30-foldenrichment. In the secondary screen, approximately 60% of the cloneswere positive with the subtracted probe but not the target probe. Of the94 clones selected from this screen, 53 were clones of mRNAs selectivelyexpressed in hypothalamus. These 53 clones correspond to approximately1% of the clones examined in this pilot study, and represented 16distinct mRNA species, suggesting that a complete characterization ofhypothalamus mRNAs might reveal 100-200 species that were specific to orhighly enriched in the hypothalamus. Of the 16 mRNA species detectedhere, 9 corresponded to already known proteins, among them oxytocin,vasopressin and POMC, three neuropeptides known to be highly enriched inthe hypothalamus. However, 7 mRNA species were novel. Among mRNA speciesnot detected in the 94-clone sample were those encoding the releasingfactors, which are less abundant than most of the species detected here.

[0218] Oxytocin and vasopressin mRNAs are predominantly associated withdiscrete hypothalamic nuclei, as was previously known. The in situhybridization images indicate that several additional mRNAs, includingseveral novel species, are enriched in the hypothalamus. Among the novelspecies, only clone 35 meets the hypothesis in its strictest sense: themRNA appears to be restricted to nuclei in the paraventricular area ofthe hypothalamus.

[0219] Other mRNAs corresponding to novel clones exhibit enrichment inbasal diencephalic structures, especially the hypothalamus, but withinthe hypothalamus none is restricted to a single nucleus. These speciespresumably encode proteins whose functions are not dedicated to singlephysiological systems. Nevertheless, their roles seem to have selectiveutility within the CNS. Previous studies looking at mRNAs enriched inthe caudate revealed several involved in signal transduction pathways(Usui, et al., supra). That is not the finding for thehypothalamus-enriched species encountered thus far.

[0220] The data suggest that the hypothalamus utilizes at least twodifferent strategies for employing selectively expressed proteins. Somespecific mRNAs are discretely correlated to distinct nuclei. Thus far,all of these mRNAs encode secretory signalling proteins. A class ofmRNAs have also been recognized that are expressed prominently inhypothalamus and amygdala. These do not appear to be restricted tofunctionally discrete regions, but their comparable anatomicalrestrictions suggest that they might participate in a series ofbiochemical processes that are selectively distributed to these regions,which are developmentally related. Thus these regions may sharemolecular properties that are not apparent at the anatomical level.

[0221] DNA sequence analysis of the complete 569 nucleotide rat clone 35revealed that the clone mRNA encodes a 130-residue putative secretoryprotein (called H35) or hypocretin with 4 sites for potentialproteolytic maturation (FIG. 5). Several proteolytic fragments have beenidentified, some replacing C-terminal glycines with amide groups. Two ofthe products of proteolysis have 14 amino acid identities across 20residues. This region of H135 includes a 7/7 match with a region of thegut hormone secretin, suggesting that the prepropeptide gives rise totwo peptide products that are structurally related both to each otherand to secretin.

[0222] The mouse homolog of clone 35 was also isolated and sequenced(FIG. 5). The mouse nucleotide sequence differs in 35 positions relativeto the rat sequence and contains 16 additional nucleotides near its 3′end. Of these differences, 19 nucleotides differ within the proteincoding region. Only 7 of these affect the encoded protein sequence. Oneamino acid difference is a neutral substitution in the secretion signalsequence (residue 3). The remaining 6 differences are in the C-terminalregion. One of these obliterates a potential proteolytic cleavage site.This observation and the nature of the other differences make itunlikely that 2 of the possible maturation products of the ratpreproprotein are functional. However, the 2 peptides that are relatedboth to each other and to secretin are absolutely preserved betweenspecies, providing strong support for the notion that these peptideshave a function conserved during evolution.

[0223] The cells that, express this mRNA are distributed in abilaterally symmetrical pattern in a previously uncharted nucleus of therat dorsal-lateral hypothalamus and sparse ependymal cells that line theventricles suggesting that the peptides function as intercellularmessengers within the CNS. Colocalization studies suggest a partialoverlap with cells positive for galanin, bradykinin and dynorphin. Therat H35 mRNA is restricted to the CNS in the studies performed to date.It is not expressed at high concentrations in immature animals.

[0224] These observations, along with the sequence data discussed above,suggest that the H35 peptides are secreted into the CSF and locallywithin the hypothalamus; that their functions are only manifested inmature animals; and that their expression is coupled to the generalhomeostatic status of the animal, although not regulated in anall-or-none fashion by homeostasis. In other words, these are newhormones that act within the central nervous system.

[0225] The polypeptides may be expressed by transformation of a suitablehost cell with a cDNA in a suitable expression vector. The choice ofhost cell is not critical. The polypeptide may be produced from aprocaryotic (e.g. E. coli) or eucaryotic (mammalian, e.g. COS-7, CHO,NIH 3T3) host cell, as desired.

[0226] The hypocretin polypeptides, and fragments thereof, of thisinvention are useful in diagnosis and therapy. Recombinant or naturalpolypeptides may be used in Western blot, ELISA, RIA, and the like, andin receptor binding assays, for direct or competitive binding studies toidentify hyprocretin specific receptors. The identification ofhypocretin analogs and antagonists is also accomplished via use of thepolypeptides identified herein. Further details of such uses aredescribed in U.S. Pat. No. 5,242,798, incorporated herein by reference.

[0227] In another aspect, the polypeptides of this invention may be usedto generate antibodies. Methods of preparing polyclonal antibodies arewell known in the art. For example, an immunogenic conjugate comprisingthe hypocretin protein or a fragment thereof, optionally linked to acarrier protein, is used to immunize a selected mammal (mouse, rabbit,et al.). Serum from the immunized mammal is collected and treated toseparate the immunoglobulin fraction. Monoclonal antibodies are preparedby standard hybridoma cell technology (Koller and Milstein, Nature256:495-497 (1975)). Briefly, spleen cells are obtained from a hostanimal immunized with an hypocretin protein or fragment. Hybrid cellsare formed by fusing these spleen cells with an appropriate myeloma cellline and cultured. The antibodies produced are screened for theirability to bind H35 by, for example, ELISA. The cells producing thehypocretin antibody are selected.

[0228] Antibodies directed to a conserved epitope common to thehypocretin polypeptides of several species will detect hypocretinpolypeptides of mammalian species in general. For example, antibodiesdirected against such a conserved sequence as GNHAAGILT (FIG. 5) can beused to detect human hypocretin polypeptides.

[0229] The polynucleotides and polypeptides of this invention may alsobe formulated into diagnostic and therapeutic compositions.Representative methods of formulation may be found in Remington: TheScience and Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton,Pa. (1995). The selection of the precise concentration, composition, anddelivery regimen is influenced by, inter alia, the specificpharmacological properties of the selected compound, the intended use,the nature and severity of the condition being treated or diagnosed, andthe physical condition and mental acuity of the intended recipient. Suchconsiderations are within the purview of the skilled artisan.

[0230] Representative delivery regimens include oral, parenteral(subcutaneous, intramuscular, and intravenous), rectal, buccal,pulmonary, transdermal, and intranasal, preferably intravenous. Thecomposition may be in solid, liquid, gel, or aerosol form. Generally,the compound will be present in an amount from about 1 μg to about 100μg, in a sterile aqueous solution, optionally including stabilizers andthe like.

[0231] The present invention also describes a diagnostic system,preferably in kit form, for assaying for the presence of a hypocretin ofthis invention in a body sample, such brain tissue, cell suspensions ortissue sections, or body fluid samples such as CSF, blood, plasma orserum, where it is desirable to detect the presence, and preferably theamount, of a hypocretin protein in the sample according to thediagnostic methods described herein.

[0232] In a related embodiment, a nucleic acid molecule can be used as aprobe (an oligonucleotide) to detect the presence of a gene or mRNA in acell that is diagnostic for the presence or expression of a hypocretinin the cell. The nucleic acid molecule probes were described in detailearlier.

[0233] The diagnostic system includes, in an amount sufficient toperform at least one assay, a subject hypocretin polypeptide, a subjectantibody or monoclonal antibody, and a subject nucleic acid moleculeprobe of the present invention, as a separately packaged reagent.

[0234] Another embodiment is a diagnostic system, preferably in kitform, for assaying for the presence of a hypocretin polypeptide oranti-hypocretin antibody in a body fluid sample such as for monitoringthe fate of therapeutically administered hypocretin polypeptide oranti-hypocretin antibody. The system includes, in an amount sufficientfor at least one assay, a subject hypocretin polypeptide and a subjectantibody as a separately packaged immunochemical reagent.

[0235] Instructions for use of the packaged reagent(s) are alsotypically included.

[0236] As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic (e.g., polyethylene, polypropylene orpolycarbonate), paper, foil and the like capable of holding within fixedlimits a polypeptide, polyclonal antibody or monoclonal antibody of thepresent invention. Thus, for example, a package can be a glass vial usedto contain milligram quantities of a hypocretin polypeptide or antibodyor it can be a microliter plate well to which microgram quantities of acontemplated polypeptide or antibody have been operatively affixed,i.e., linked so as to be capable of being immunologically bound by anantibody or antigen, respectively.

[0237] “Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent or sample admixtures,temperature, buffer conditions and the like.

[0238] A diagnostic system of the present invention preferably alsoincludes a label or indicating means capable of signaling the formationof an immunocomplex containing a polypeptide or antibody molecule of thepresent invention.

[0239] The word “complex” as used herein refers to the product of aspecific binding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

[0240] As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents. Such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and systems.

[0241] The labeling means can be a fluorescent labeling agent thatchemically binds to antibodies or antigens without denaturing them toform a fluorochrome (dye) that is a useful immunofluorescent tracer.Suitable fluorescent labeling agents are fluorocbromes such asfluorescein isocyanate (FIC), fluorescein isothiocyante (FITC),5-diethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis, etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

[0242] In preferred embodiments, the indicating group is an enzyme, suchas horseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-amino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

[0243] Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which themselvesemit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Cr represent oneclass of gamma ray emission-producing radioactive element indicatinggroups. Particularly preferred is ¹²⁵I. Another group of useful labelingmeans are those elements such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N which themselvesemit positrons. The positrons so emitted produce gamma rays uponencounters with electrons present in the animal's body. Also useful is abeta emitter, such as ¹¹¹In or ³H.

[0244] The linking of labels, i.e., labeling of, polypeptides andproteins is well known in the art. For instance, antibody moleculesproduced by a hybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:346(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795.

[0245] The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

[0246] In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

[0247] The diagnostic kits of the present invention can be used in an“ELISA” format to detect the quantity of hypocretin in a sample. “ELISA”refers to an enzyme-linked immunosorbent assay that employs an antibodyor. antigen bound to a solid phase and an enzyme-antigen orenzyme-antibody conjugate to detect and quantify the amount of anantigen present in a sample. A description of the ELISA technique isfound in Chapter 22 of the 4th Edition of Basic and Clinical Immunologyby D. P. Sites et al., published by Lange Medical Publications of LosAltos, Calif. in 1982 and in U.S. Pat. No. 3,654,090; U.S. Pat. No.3,850,752; and U.S. Pat. No. 4,016,043, which are all incorporatedherein by reference.

[0248] In some embodiments, a hypocretin polypeptide, an antibody or amonoclonal antibody of the present invention can be affixed to a solidmatrix to form a solid support that comprises a package in the subjectdiagnostic systems.

[0249] A reagent is typically affixed to a solid matrix by adsorptionfrom an aqueous medium although other modes of affixation applicable toproteins and polypeptides can be used that are well known to thoseskilled in the art. Exemplary adsorption methods are described herein.

[0250] Useful solid matrices are also well known in the art. Suchmaterials are water insoluble and include the cross-linked dextranavailable under the trademark SEPHADEX from Pharmacia Fine Chemicals(Piscataway, N.J.); agarose; beads of polystyrene beads about 1 micron(μ) to about 5 millimeters (mm) in diameter available from AbbottLaboratories of North Chicago, Ill.; polyvinyl chloride, polystyrene,cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such assheets, strips or paddles; or tubes, plates or the wells of a microliterplate such as those made from polystyrene or polyvinylchloride.

[0251] The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microliter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

[0252] The packaging materials discussed herein in relation todiagnostic systems are those customarily utilized in diagnostic systems.

[0253] G. Cell Lines Expressing Hypocretin

[0254] The invention also includes a host cell transformed with arecombinant DNA (recombinant DNA) molecule of the present invention. Thehost cell can be either procaryotic or eucaryotic, although eucaryoticcells are preferred, particularly mammalian cells. Preferred cells areisolated, that is, substantially homogeneous and therefore free fromother cell types or other cells having a hypocretin protein expressedtherein.

[0255] A cell expressing a hypocretin of this invention has a variety ofuses according to this invention. Particularly preferred are uses forbulk production of hypocretin, for the purpose of providing immunogenfor production of antibody, for supply of therapeutic protein, fordirect binding or for screening pharmaceutical compound banks for thepresence of hypocretin receptor-specific ligands, i.e., in drugscreening assays as described herein. Thus, particularly preferred arecells containing a recombinant DNA molecule that expresses a hyppcretinprotein of this invention.

[0256] In one embodiment, a cell is produced for transplantation into abody tissue, thereby expressing hypocretin and providing replacementtherapy. The cell can be syngeneic, and typically will be a braintissue-derived cell, such as a hippocampal cell, neonatal brain tissuecell, glioma and the like neuronal tissue cell. Transplantation isaccomplished using surgical procedures available to a neurosurgeon wherethe transplantation is to be made into the brain, brain stem or otherneurological tissues. In preferred embodiments, the cell contains avector for expressing the hypocretin in which the expression means isunder the control of a regulatable promoter, as is well known, such thatexpression of the hypocretin protein can be regulated.

[0257] Eucaryotic cells useful for expression of a hypocretin proteinare not limited, so long as the cell or cell line is compatible withcell culture methods and compatible with the propagation of theexpression vector and expression of the hypocretin protein gene product.Preferred eucaryotic host cells include yeast and mammalian cells,preferably vertebrate cells such as those from a mouse, rat, monkey orhuman fibroblastic cell line. Preferred eucaryotic host cells includeChinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIHSwiss mouse embryo cells NIH/3T3 (ATCC CRL 1658), HELA cells (ATCC CCL2), baby hamster kidney cells (BHK), COS-7, COS-1, HEK293 (ATCC CRL1573), Ltk-1, AV-12 (ATCC CRL 9595), and the like eucaryotic tissueculture cell lines.

[0258] Transformation of appropriate cell hosts with a recombinant DNAmolecule of the present invention is accomplished by well known methodsthat typically depend on the type of vector used. With regard totransformation of procaryotic host cells, see, for example, Cohen etal., Proc. Natl. Acad. Sci. USA, 69:2110 (1972); and Manlatis et al.,Molecular Cloning, A Laboratory Mammal, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982).

[0259] With regard to transformation of vertebrate cells with vectorscontaining recombinant DNAs, see, for example, Graham et al., Virol.,52:456 (1973); Wigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76(1979), and the teachings herein.

[0260] Successfully transformed cells, i.e., cells that contain arecombinant DNA molecule of the present invention, can be identified bywell known techniques. For example, cells resulting from theintroduction of an recombinant DNA of the present invention can becloned to clonally homogeneous cell populations that contain therecombinant DNA. Cells from those colonies can be harvested, lysed andtheir DNA content examined for the presence of the recombinant DNA usinga method such as that described by Southern, J. Mol. Biol., 98:503(1975) or Berent et al., Biotech., 3:208 (1985).

[0261] In addition to directly assaying for the presence of recombinantDNA, successful transformation can be confirmed by well knownimmunological methods when the recombinant DNA is capable of directingthe expression of hypocretin or by the detection of hypocretin bindingactivity.

[0262] For example, cells successfully transformed with an expressionvector produce proteins displaying hypocretin antigenicity or biologicalactivity. Samples of cells suspected of being transformed are harvestedand assayed for either hypocretin biological activity or antigenicity.

[0263] In addition to the transformed host cells themselves, the presentinvention also includes a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium. Preferably, the culture alsocontains a protein displaying hypocretin antigenicity or biologicalactivity.

[0264] Nutrient media useful for culturing transformed host cells arewell known in the art and can be obtained from several commercialsources. In embodiments wherein the host cell is mammalian, a“serum-free” medium can be used.

[0265] H. Screening Methods to Identify Agonists and Antagonists ofHypocretin

[0266] The ability to selectively bind/modulate function of a hypocretinreceptor by a hypocretin ligand is at the heart of useful hypocretinpharmacology, and depends on identifying pharmacological molecules whichcan act a selective ligands, agonists or antagonists for a hypocretinreceptor. To that end, the elucidation of new hypocretin proteins, suchas those described herein, provides valuable tools for the search forselective reagents, tools that are useful in binding assays, and inscreening assays which indicate selective drug response to thehypocretin receptor.

[0267] The invention includes methods for determining whether a moleculebinds to, and preferably whether the molecule activates, a preselectedhypocretin receptor.

[0268] The method comprises conducting a binding assay to identifymolecules which bind the hypocretin receptor, as described in any of theassays herein. Thus, the method comprises (1) contacting a candidatemolecule with a cell having a hypocretin receptor under conditionspermitting binding of hypocretin to the receptor, and (2) detecting thepresence of the candidate molecule bound to the hypocretin receptor,thereby determining whether the candidate binds to the receptor. Thereceptor is typically a cell surface protein when expressed by thecells.

[0269] Alternatively, one can use a competition format to identifyanalogs of hypocretin by using a labeled hypocretin, and measuring theamount of bound label in the presence of a candidate ligand, indicatingwhether the candidate competes with labeled hypocretin for binding tothe receptor. An exemplary competition assay is described herein.

[0270] It is also possible to use the above method to determine whetherthe molecule which binds to the hypocretin receptor also activates ormotivates the receptor's function, i.e., acts as an agonist, ordetermine whether the molecule inhibits the receptor's function, i.e.,acts as an antagonist, or acts as and inverse agonist. Thus, byevaluating in the detecting step whether the hypocretin receptor isactivated, one determines whether the candidate molecule is bioactive.

[0271] Methods for detecting bioactivity of the candidate molecule canvary, but typically involve measuring changes in intracellular levels ofa secondary messenger effected as a result of binding, detecting changesin electrical potential, observing physiological or behavioral effectsrelated to hypocretin function, and the like methods. Exemplary assaysfor binding or for hypocretin-specific bioactivity are described in theExamples and include measurement of electrical changes of hypothalamicneurons, measurement of food intake or body temperature, or directbinding to a cell having a hypocretin receptor.

[0272] It is noted that the hypocretin receptor has not beencharacterized in extensive detail. Thus, any receptor that bindshypocretin can be referred to as a hypocretin receptor for the purposesof a screening assay, although receptors with the highest affinity andspecificity for hypocretin are preferred. In practicing the presentscreening methods, one can use any of a variety of cells lines ortissues that possess a hypocretin receptor, including the exemplary celllines and tissues described herein. The invention should not beconstrued as limiting so long as the binding or bioactivity assayinvolves the use of a hypocretin receptor. In preferred embodiments, areceptor that is specific for hypocretin should be used. Specificity canbe demonstrated by well known methods of ligand binding andligand-mediated activation.

[0273] A related embodiment includes a method for screening to identifya candidate molecule that can bind, inhibit or activate a preselectedhypocretin receptor by functioning as a hypocretin agonist orantagonist. The method comprises:

[0274] (a) contacting a mammalian cell with said candidate drug underconditions permitting activation of said hypocretin receptor byhypocretin; and

[0275] (b) detecting the activation status of said hypocretin receptor,and thereby determining whether the drug activates or inhibits saidreceptor.

[0276] I. Methods for Altering Hypocretin Receptor Function

[0277] a. Therapeutic Methods

[0278] The certain reagents described in the present invention have thecapacity to modulate hypocretin receptor function, such as agonists orantagonists, and therefore are useful in therapeutic methods forconditions mediated by the hypocretin receptor.

[0279] Hypocretin polypeptides that mimic exposed regions of hypocretinhave the ability to function as analogs and compete for binding to thehypocretin receptor, or for other agents that would normally interactwith the receptor, thereby inhibiting binding of hypocretin to thereceptor.

[0280] Furthermore, antibodies and monoclonal antibodies of the presentinvention that bind to exposed regions of hypocretin have the capacityto alter hypocretin receptor function by blocking natural interactionswith hypocretin that normally interact at the site. Exemplary antibodiesare the anti-hypocretin antibodies described earlier.

[0281] Finally, oligonucleotides are described herein which arecomplementary to mRNA that encodes a hypocretin protein of thisinvention and that are useful for reducing gene expression andtranslation of the hypocretin mRNA, thereby altering hypocretin levelsin a tissue.

[0282] In one embodiment, the present invention provides a method formodulating hypocretin function in an animal or human patient comprisingadministering to the patient a therapeutically effective amount of aphysiologically tolerable composition containing a hypocretinpolypeptide, analog or peptidomimetic, anti-hypocretin antibody ormonoclonal antibody, hypocretin agonist or antagonist, or anoligonucleotide of the present invention.

[0283] A therapeutically effective amount of a hypocretin polypeptide,as an example for practicing the invention, is a predetermined amountcalculated to achieve the desired effect, i.e., to modulate receptorinteraction with its normal target, and thereby interfere with normalreceptor function. Depending on the structure of the particular peptidethe binding of some peptides will activate the receptor, while bindingof other peptides will not activate the receptor.

[0284] Similarly, a therapeutically effective amount of ananti-hypocretin antibody is a predetermined amount calculated to achievethe desired effect, i.e., to immunoreact with the hypocretin, andthereby inhibit the hypocretin receptor's ability to interact with itsnormal target, hypocretin, and thereby interfere with normal receptorfunction.

[0285] The in vivo inhibition of hypocretin receptor function using ahypocretin polypeptide, an anti-hypocretin antibody, or hypocretinagonist or antagonist of this invention is a particularly preferredembodiment and is desirable in a variety of clinical settings, such aswhere the patient is exhibiting symptoms of an over or under activatedhypocretin receptor.

[0286] A therapeutically effective amount of a hypocretin polypeptide,agonist or antagonist of this invention is typically an amount such thatwhen administered in a physiologically tolerable composition issufficient to achieve a plasma concentration of from about 0.1 nanomolar(nM) to about 100 nM, and preferably from about 0.5 nM to about 10 nM.

[0287] A therapeutically effective amount of an antibody of thisinvention is typically an amount of antibody such that when administeredin a physiologically tolerable composition is sufficient to achieve aplasma concentration of from about 0.1 microgram (μg) per milliliter(ml) to about 100 μg/ml, preferably from about 1 μg/ml to about 5 μg/ml,and usually about 5 μg/ml.

[0288] The effectiveness of the therapy can be determined by observingablation of the symptoms associated with the function of the hypocretinreceptor being inhibited.

[0289] The therapeutic compositions containing a hypocretin polypeptide,agonist, antagonist or anti-hypocretin antibody of this invention areconventionally administered intravenously or by a method for delivery toa brain tissue, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

[0290] Delivery to a brain tissue or CSF can be accomplished by avariety of means, including by direct injection, by use of a cannulainto the target tissue, by direct application in a surgical procedure,by adsorption across the blood-brain barrier following intravenousadministration, by viral vectors, and the like means.

[0291] The therapeutic compounds and compositions are generallyadministered so as to contact the cells or the tissue containing cellswhich contain the target hypocretin receptor. This administration can beaccomplished by introduction of the composition internally such asorally, intravenously, intramuscularly, intranasally or via inhalationof aerosols containing the composition, and the like, by cannula into abrain tissue, or by introduction into or onto a tissue system as byintroduction transdermally, topically or intralesionally, insuppositories, or by intra-orbital injection, and the like.

[0292] The compositions are administered in a manner compatible with thedosage formulation, and in a therapeutically effective amount. Thequantity to be administered depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgement of thepractitioner and are particular to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the CSF or blood in the ranges specified for in vivotherapies are included.

[0293] As an aid to the administration of effective therapeutic amountsof a hypocretin polypeptide, agonist, antagonist, antibody, ormonoclonal antibody, (hereinafter a “therapeutic agent”) a diagnosticmethod of this invention for detecting a therapeutic agent in thesubject's CSF or blood is useful to characterize the fate of theadministered therapeutic agent. Suitable diagnostic (monitoring) assaysare described herein.

[0294] b. Methods for Inhibiting Gene Expression

[0295] In another embodiment, the invention includes the use of nucleicacids encoding portions of a hypocretin gene for inhibiting geneexpression and function.

[0296] The present invention provides for a method for inhibitingexpression of hypocretin gene products and thereby inhibiting thefunction of the target hypocretin protein. The DNA segments and theircompositions have a number of uses, and may be used in vitro or in vivo.In vitro, the compositions may be used to block function and expressionof hypocretin in cell cultures, tissues, organs and the like materialsthat can express hypocretin. In vivo, the compositions may be usedprophylactically or therapeutically for inhibiting expression of ahypocretin gene, and by inhibiting diseases or medical conditionsassociated with the expression or function of the hypocretin gene or theactivity state of its receptor.

[0297] The method comprises, in one embodiment, contacting cells ortissues with a therapeutically effective amount of a pharmaceuticallyacceptable composition comprising a DNA segment of this invention. In arelated embodiment, the contacting involves introducing the DNA segmentcomposition into cells expressing a hypocretin protein.

[0298] The DNA segment can be in a variety of forms, but is preferablyin a single-stranded form to facilitate complementary hybridization tothe target mRNA in the cell in which the hypocretin gene expression isto be altered.

[0299] The term “cells” is intended to include a plurality of cells aswell as single cells. The cells can be isolated, or can be cells thatform a larger organization of cells to form a tissue or organ.

[0300] Another embodiment is a method of inhibiting the expression ofhypocretin genes in a patient comprising administration to the patientof a therapeutically effective amount of a DNA segment composition ofthis invention in a pharmaceutically acceptable excipients. In caseswhere the distribution of the hypocretin is believed to be disseminatedin the body, the administration of therapeutic oligonucleotide can besystemic. Alternatively, the target hypocretin can be localized to atissue, and the therapeutic method can likewise be directed atdelivering the therapeutic DNA segment to the tissue to be treated.

[0301] The concentration of the active DNA segment ingredient in atherapeutic composition will vary, depending upon the desired dosage,use, frequency of administration, and the like. The amount used will bea therapeutically effective amount and will depend upon a number offactors, including the route of administration, the formulation of thecomposition, the number and frequency of treatments and the activity ofthe formulation employed.

[0302] The use of therapeutic DNA segments, and therefore the deliveryof those DNA segments into cells where they are effective, has beendescribed in a variety of settings. It is generally known thattherapeutically effective intracellular levels of nucleic acids, andparticularly smaller nucleic acids such as DNA segments andoligonucleotides, can be achieved by either exposing cells to solutionscontaining nucleic acids or by introduction of the nucleic acids intothe inside of the cell. Upon exposure, nucleic acids are taken up by thecell where they exert their effectiveness. In addition, directintroduction into the cell can be provided by a variety of means,including microinjection, delivery by the use of specific uptakevehicles, and the like.

[0303] The pharmaceutical composition containing the therapeuticoligonucleotide preferably also contains physiologically acceptablecarriers, in particular hydrophobic carriers which facilitate carryingthe oligonucleotide through the cell membrane or blood brain barrier.

[0304] Exemplary descriptions of the delivery of therapeutic DNAsegments and oligonucleotides into cells can be found in the teachingsof U.S. Pat. Nos. 5,04,820, 4,806,463, 4,757,055, and 4,689,320, whichteachings are hereby incorporated by reference.

[0305] A therapeutically effective amount is a predetermined amountcalculated to achieve the desired effect, i.e., to bind to a hypocretingene present and thereby inhibit function of the gene.

[0306] As is apparent to one skilled in the art, the copy number of ahypocretin gene may vary, thereby presenting a variable amount of targetwith which to hybridize. Thus it is preferred that the therapeuticmethod achieve an intracellular concentration of a therapeutic DNAsegment of this invention in molar excess to the copy number of the genein the cell, and preferably at least a ten-fold, more preferably atleast a one-hundred fold, and still more preferably at least a onethousand-fold excess of therapeutic DNA segments relative to the genecopy number per cell. A preferred effective amount is an intracellularconcentration of from about 1 nanomolar (nM) to about 100 micromolar(μM), particularly about 50 nM to about 1 μM.

[0307] Alternatively, a therapeutically effective amount can beexpressed as an extracellular concentration. Thus it is preferred toexpose a cell containing a hypocretin gene to a concentration of fromabout 100 nM to about 10 millimolar (mM), and preferably about 10 μM to1 mM. Thus, in embodiments where delivery of a therapeutic DNA segmentcomposition is designed to expose cells to the nucleic acid for cellularuptake, it is preferred that the local concentration of the DNA segmentin the area of the tissue to be treated reach the extracellularconcentrations recited above.

[0308] For patient dosages, using a 20 nucleotide base double-strandedDNA segment as the standard, a typical dosage of therapeutic compositionfor a 70 kilogram (kg) human contains in the range of about 0.1milligram (mg) to about 1 gram of 20-mer DNA segment per day, and moreusually in the range of about 1 mg to 100 mg per day. Stateddifferently, the dosage is about 1 μg/kg/g day to about 15 mg/kg/day,and preferably about 15 to 1500 μg/kg/day.

[0309] The in vivo inhibition of hypocretin gene expression and functionby a therapeutic composition of this invention is desirable in a varietyof clinical settings, such as where the patient is at risk for diseasebased on expression of the hypocretin gene.

[0310] c. Therapeutic Compositions

[0311] The present invention includes therapeutic compositions usefulfor practicing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with a therapeutic reagent of this invention,namely a hypocretin polypeptide, an anti-hypocretin antibody ormonoclonal antibody, or oligonucleotide as described herein, dissolvedor dispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not immunogenic when administered to amammal or human patient for therapeutic purposes.

[0312] As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

[0313] The preparation of a pharmacological composition that containsactive ingredients dissolved or dispersed therein is well understood inthe art. Typically such compositions are prepared as injectables eitheras liquid solutions or suspensions, however, solid forms suitable forsolution, or suspensions, in liquid prior to use can also be prepared.The preparation can also be emulsified.

[0314] The active ingredient can be mixed with excipient which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipient are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

[0315] The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

[0316] Physiologically tolerable carriers are well known in the art.Exemplary of liquid carriers are sterile aqueous solutions that containno materials in addition to the active ingredients and water, or containa buffer such as sodium phosphate at physiological pH value,physiological saline or both, such as phosphate-buffered saline. Stillfurther, aqueous carriers can contain more than one buffer salt, as wellas salts such as sodium and potassium chlorides, dextrose, polyethyleneglycol and other solutes.

[0317] As described herein, for intracellular delivery ofoligonucleotides, specialized carriers may be used which facilitatetransport of the oligonucleotide across the cell membrane. Thesetypically are hydrophobic compositions, or include additional reagentswhich target delivery to and into cells.

[0318] Liquid compositions can also contain liquid phases in addition toand to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions.

[0319] A therapeutic composition contains an amount of a hypocretinpolypeptide or anti-hypocretin antibody molecule of the presentinvention sufficient to inhibit hypocretin function. Typically this isan amount of at least 0.1 weight percent, and more preferably is atleast 1 weight percent, of peptide or antibody per weight of totaltherapeutic composition. A weight percent is a ratio by weight ofpeptide or antibody to total composition. Thus, for example, 0.1 weightpercent is 0.1 grams of polypeptide per 100 grams of total composition.

[0320] The following Examples are illustrative of one means ofpracticing certain aspects of the invention disclosed herein and shouldnot be construed so as impart any undue limitations upon the inventionas claimed below.

EXAMPLE 1

[0321] Young adult Sprague-Dawley rats of both genders were sacrificedunder anesthesia by decapitation and their brains quickly removed. Thehypothalamus, hippocampus, and cerebellum were immediately dissected onan ice-cold plate following the boundaries described by Glowinski andIversen (Glowinski, J., & Iversen, L. L., J. Neurochem. 13:655-669,1966). The block of hypothalamic tissue was 2 mm deep and was takenusing the optic chiasm as the rostral limit and the mammillary bodies ascaudal reference. Cytoplasmic RNA was isolated rapidly from thedissected tissues (Schibler, K., Tosi, M., Pittet, A. C., Fabiani, L., &Wellauer, P. K., J. Mol. Biol. 142:93-116, 1980) and enriched forpoly(A)-containing species by oligo(dT)-cellulose chromatography (Aviv,H., & Leder, P., Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972). For theNorthern blots, RNA was isolated (Chirgwin, J. M., Przybyla, A. E.,MacDonald, R. J., & Rutter, W. J., Biochemistry 18:5294, 1979) fromfrozen tissue purchased from Zivic-Miller Zelienople, Pa.). cDNAlibraries were prepared as described previously Usui et al, supra,except that pBCSK⁺ was used for the subtracted library rather thanpT7T3D because lower backgrounds have been found in the subsequent stepsusing the former vector (H. Usui, personal communication). The number ofrecombinants in the libraries were: pT7T3D hypothalamus 8×10⁶;cerebellum pGEM11Zf (−) 5×10⁵; hippocampus pGEM11Zf(−) 1×10⁶.

[0322] Subtractive hybridization was performed in two cycles using thepreviously described procedure (Usui et al., supra). Briefly, 1 μg oftrace-labeled, tagged hypothalamus target cDNA prepared as describedfrom the pT7T3D target library was annealed for 24 hrs at 68 degreeCelsius in 10 μg of hybridization buffer (Usui et al, supra) with 20 μgcerebellum cRNA (ratio 1:20). After hydroxyapatite chromatography, thesingle-stranded fraction corresponded to 10% of the input material, asjudged by tracer quantitation. This was mixed with 20 μg of hippocampuscRNA (estimated ratio 1:200) for a second 24 hr hybridization, afterwhich 30% of the input chromatographed at the single-strand position.Cumulatively, these steps removed more than 97% of the input tracer. Analiquot of the single-stranded material was used as template in a30-cycle PCR (program: 94 degree Celsius for 15 sec, 60 degree Celsiusfor 15 sec, 72 degree Celsius for 1 min) using primers corresponding tothe tag sequences (Usui et al., supra): 5′-AACTGGAAGAATTCGCGG-3′ and5′-AGGCCAAGAATTCGGCACGA-3′. The amplification product was cleaved withNotI, then EcoRI, and inserted into pBCSK⁺. A dot blot was prepared andscreened with probes prepared from the target, subtracted target anddriver libraries as previously described by Usui et al, supra, usingserial dilutions of plasmid cDNA clones isolated previously in thislaboratory. The target and subtracted target cDNA libraries werescreened to determine the frequency of oxytocin and VAT-1 cDNA clonesusing as probes clones isolated in the present study.

[0323] Clone 35 cDNA from the subtracted rat hypothalamus library wasused as a probe to screen a rat brain cDNA library in the plasmid pHG327as described by Forss-Petter et al., J. Mol. Neurosci. 1:63-75 (1989).The cDNA library was constructed as described by Staeheli et al., Cell44:147-158 (1986).

EXAMPLE 2

[0324] Similarly, following the procedures of Example 1, a mouse(C57/B16) hypothalamus cDNA library, constructed in the pT7T3D vector,was used as a template for PCR amplification (primers 5′TAAGACGACGGCCTCAG 3′ and 5′ CACACCAACAGAGAAACG 3′) to obtain the mousehomolog of the rat H35 cDNA obtained above. The mouse and rat cDNA andprotein sequences are compared in FIG. 5. The 569 nucleotide ratsequence has the potential to encode a 130-residue putative secretoryprotein (preprohypocretin) with an apparent signal sequence and 3additional sites for potential proteolytic maturation (FIG. 5A). Two ofthe putative products of proteolysis (hcrt1 and hcrt2) have 14 aminoacid identities across 20 residues (FIG. 5B). This region of one of thepeptides contains a 7/7 match with secretin (FIG. 5B), suggesting thatthe prepropeptide gives rise to two peptide products that arestructurally related both to each other and to secretin.

[0325] The mouse hypocretin nucleotide sequence differs in 35 positionsrelative to the rat, and contains 16 additional nucleotides near its 3′end. Of these differences, 19 are within the putative protein-codingregion (FIG. 5A), only 7 of which affect the encoded protein sequence:one amino acid difference at residue 3 is a neutral substitution in theapparent secretion signal sequence; the remaining 6 differences are nearthe C-terminus, one of which obliterates a potential proteolyticcleavage site. The absence of this site and the nature of the otherdifferences make it unlikely that two of the four possible ratmaturation products are generated and functional in mice. However, thetwo putative hcrt peptides that are related both to each other and tothe secretin family are absolutely preserved between the two species,providing strong support for the notion that these peptides have afunction conserved during evolution. Both hcrt1 and hcrt2 terminate withglycine residues, which typically are substrates for, leaving thenitrogen of the terminal glycine as a C-terminal amide in the maturepeptide.

[0326] Several hypocretin peptides are distinguished within the sequenceof hypocretin (FIG. 5). The peptide from about amino acid residue 28 toabout amino acid residue 130 (SEQ ID NO: 6) represents the peptideproduced by cleavage of the signal peptide. The peptide from about aminoacid residue 28 to about amino acid residue 66 (SEQ ID NO: 7)corresponds to hcrt1. The peptide from about amino acid residue 28 toabout amino acid residue 65 (SEQ ID NO: 8) corresponds to hcrt1 maturedby peptidylglycine alpha-amidating monooxygenase, leaving the nitrogenof the terminal glycine as a C-terminal amide in the mature peptide. Thepeptide from about amino acid residue 70 to about amino acid residue 97(SEQ ID NO: 9) corresponds to hcrt2. The peptide from about amino acidresidue 70 to about amino acid residue 96 (SEQ ID NO: 10) corresponds tohcrt2 matured by peptidylglycine alpha-amidating monooxygenase, leavingthe nitrogen of the terminal glycine as a C-terminal amide in the maturepeptide. The peptide from about amino acid residue 47 to about aminoacid residue 66 (SEQ ID NO: 11) corresponds to the consensus sequenceregion of hcrt1 (FIG. 5B). The peptide from about amino acid residue 78to about amino acid residue 97 (SEQ ID NO: 12) corresponds to theconsensus region of Hrct2. The peptide GNHAAGILT (SEQ ID NO: 13) iscommon to both hcrt1 and hcrt2.

EXAMPLE 3

[0327] Rat H35 (SEQ ID NO: 3) is inserted into the BamiH1 sites of apHG237 vector. Upon digestion with BamH1 restriction enzyme, theresultant 569 bp fragment is then inserted directly into the BglII siteof the polylinker region of the pCM 4 vector (D. Russell, U. TexasSouthwestern Medical Center, Dallas, Tex.), which uses thecytomegalovirus (CMV) promoter. Several eight to ten amino acid epitopetags are added by PCR to the C-terminus of H35 to allow visualization ofthe expressed product.

[0328] The respective 5′ and 3′ primers,ATCGAGATCTAGACACCATGAAGCTTCCTTGTACAAAGGTT 3′ and 5′ACTGTCTAGATCATAGATCTTCTTCAGAAATAAGTTTTTGTTGGACTCTG GATCCGCCCCGGGGCGCT3′,

[0329] are used as primers to amplify H35 beginning at position 85 inSEQ ID NO: 3 with an inserted BglII site added at its 5′ end to the 3′end having an inserted c-myc epitope tag. The PCR products are subclonedinto pCMV and transfected into a mammalian host cell to produce anH35-myc tagged protein product.

[0330] H35 proteins are also produced in bacteria by subcloning the H35coding sequence into pRSET B (Invitrogen, San Diego, Calif.), whichencodes six histidines prior to the H35 sequence. The vector contains aT7 promoter which drives expression of 6×His-tagged proteins in E. coli.The respective 5′ and 3′ oligonucleotides 5′ATCGAGATCTCTTGGGGTGGACGCGCAGCCT 3′ and 5′ ACTGAATTCTCAGACTCTGGATCCGCCCCG3′ are used as PCR primers to amplify the rat H35 sequence into theBglII and EcoRI sites of the pRSET B vector. The resultinghypocretin-poly-(His) fusion protein may be purified by affinitychromatography on a metal affinity resin.

[0331] An H35-glutathione-S-transferase fusion protein is produced in E.coli by subcloning the H35 sequence into a pGEX2 vector (Pharmacia).

EXAMPLE 4

[0332] The mouse Hcrt gene was mapped to Chromosome 11 using aninterspecific backcross. A single-strand sequence polymorphism betweenC57BL/6J and SPRET/Ei was detected as previously described and mapped onThe Jackson Laboratory BSS panel. An Hcrt-specific product ofapproximately 600 base pairs was amplified from mouse C57BL/6J genomicDNA using synthetic oligonucleotides 5′-GACGGCCTCAGACTTCTTGG-3′ and5′-GCAACAGTTCGTAGAGACGG-3′. This product contained a putative intron,and its identity as hcrt was confirmed by sequencing (data not shown).Genotype data and references for these and other linked markers can beaccessed via the Mouse Genome Database (http:/lwww.informaticsjax.org).

[0333] No recombinants in 94 BSS mice were found between hcrt and thepreviously mapped loci Brcal, Tubg and Mpmv8, placing Hcrt maximallywithin 3.8 cM (95% confidence limit) of these genes. The Hoxb cluster isapproximately 1 cM centromeric to Hcrt, and the Kcnj2 gene is locatedapproximately 4 cM telomeric. Hcrt is located in the portion of mouseChromosome 11 that shows conserved synteny with human Chromosome17q21-q24.

[0334] In Northern blot studies using poly(A)⁺ RNA prepared from brainand different peripheral tissues, the 700-nucleotide hypocretin mRNA wasdetected only in brain samples. Previous studies with RNA from differentregions of the brain had detected the hypocretin mRNA predominantly inhypothalamus samples. In samples of RNA from whole brains of developingrats, hypocretin mRNA was detected at low concentrations as early asembryonic day 18, but increased in concentration dramatically after thethird postnatal week. There was no detectable difference between brainsamples from adult males and females, suggesting that the late onset wasnot related to sexually dimorphic processes. In situ hybridizationstudies detected cell bodies in the dorsal-lateral hypothalamus and incells that line the ventricles.

EXAMPLE 5

[0335] A polyclonal antiserum (serum 2050) was raised to a chemicallysynthesized peptide corresponding to the C-terminal 17 amino acidresidues (CPTATATACAPRGGSRV) of the rat preprohypocretin sequence. InWestern transfer blots using as target electrophoretically separatedproteins from bacteria transformed with the plasmid pRSET B engineeredto express preprohypocretin, a single prominent immunoreactive band wasobserved with a migration of approximately 19 kDa with the hyperimmuneserum, but not with the preimmune serum. No immunoreaction was detectedwith an extract from bacteria transformed with a preprohypocretin/pRSETB expression plasmid, indicating that detection of the 19 kDa targetrequires hypocretin expression. Analogous results were obtained with anadditional antiserum to the 17 mer and two antisera to synthetic hcrt2.

[0336] In immunohistochemical studies with antiserum 2050 on sectionsfrom perfused adult male rats, immunoreactive cell bodies were observedexclusively in the perifornical nucleus and dorsal and lateralhypothalamic areas, consistent with the in situ hybridization results(FIG. 4). This coincident staining, its elimination when the serum waspreincubated with the peptide immunogen, and the very low nonspecificbackground observed, together with the Western blot results, providedstrong evidence for the specificity of the antiserum for hypocretin. Inaddition to cell bodies, the serum detected a prominent network offibers located within the hypothalamus, particularly the posteriorregion. Less prominent fiber projections were observed in apparentterminal fields within the preoptic area, the medial dorsal and reuniensnuclei of the thalamus, the dorsal raphe nucleus, the locus coeruleus,the laterodorsal tegmental nucleus, the central gray, the colliculi andthe nucleus of the solitary tract. In immuno-electron microscopystudies, immunoreactive secretory vesicles were observed.

EXAMPLE 6

[0337] The putative structures of the hypocretins, their expressionwithin the dorsolateral hypothalamus and accumulation within fibers andvesicles suggested that they may have intercellular signaling activity.To test this hypothesis, 10-day cultures of synaptically-coupled rathypothalamic neurons were prepared and postsynaptic currents wererecorded under voltage clamp. Application of a synthetic peptidecorresponding to amidated hcrt2 at 1 μM evoked a substantial, butreversible, increase in the frequency of postsynaptic currents in 75% ofthe neurons tested (FIG. 7A), indicating an increase in the activity ofpresynaptic axons, and suggesting an increase in excitation. The other25% of the cells showed no response to hcrt2. There was little responseby hypothalamic neurons that had been in culture for only 3-5 days,suggesting that a certain degree of synaptic maturity was required forthe effect. Hcrt2 elicited no response from synaptically coupledhippocampal dentate granule neurons in culture, demonstrating targetselectivity and suggesting that specific receptors for hcrt2 may exist.

EXAMPLE 7

[0338] Synthetic, amidated hcrt2 peptide at different concentrations wasinfused intracerebroventricularly in rats and body temperature by wasmonitored telemetry. Stereotactic ablation studies have previouslyimplicated the dorsal-lateral hypothalamus in feeding behavior, bloodpressure, and central regulation of immune function, although precisenuclei have not been correlated with these activities. A threshold-typeresponse was obtained in which, at the highest dose, 10 μg, bodytemperature dropped from 37.7 to 36.7 degrees Celsius over 30 minutesfollowing administration, then recovered to normal over 2 hours. Foodintake was monitored over 2 hours following administration and a 40%reduction in food intake was measured at a dose of 5 μg. Whereas theconcentrations of peptide required for an effect might seem high, theyare comparable to the doses of leptin administered ICV to obtain acomparable suppression of food intake. The presumed target cells ofhypocretin may not be very accessible by this unphysiological mode ofadministration. Local injection or intravenous administration ofhypocretin might be more suitable for physiological studies.

[0339] The cell bodies that produce the hypocretins are located in anarea implicated in ablation studies as regulatory centers for appetitivebehaviors, suggesting that the hypocretins may serve as a majortransmitters for the central system signalling the status of energybalance in the major fat repositories. The projections ofhypocretin-producing cells indicate that the peptides function bothwithin the hypothalamus and at a complex and diffuse network of targetsin several regions of the brain that may coordinate the various aspectsof appetitive behavior, adaptive thermogenesis and metabolic regulation.

[0340] Rat hypothalamus from 18-day embryos was cultured for 10 days invitro.

[0341] The mediobasal hypothalamus was removed from embryonic day 18Sprague Dawley rats. The tissue was enzymatically digested in a mildprotease solution (10 U/ml papain and 0.2 mg/ml L-cysteine in Earle'sbalanced salt solution) for 30 minutes. Next, the tissue was pelleted,and the protease solution was removed. Tissue was then suspended instandard tissue culture medium (glutamate- and glutamate-free DMEMsupplemented with 10% fetal bovine serum, 100 U/mlpenicillin/streptomycin, and 6 gm/l glucose) and then triturated into asingle-cell suspension. Cells were washed and pelleted an additionalthree times. The single-cell suspension was plated onto 22 mm² glasscoverslips that had been coated with high-molecular-weight (540,000 Da)poly-D-lysine. High-density cultures (200,000/ cm²) were used for allexperiments. Hypothalamic neural cultures were maintained in a Napco3600 incubator (37 degree Celsius and 5% CO₂) until they were ready foruse. To limit non-neuronal cell proliferation cytosine arabinofuranoside(1 μM) was added to the tissue culture medium 1 day after plating.

[0342] Synaptically coupled hypothalamic neurons were recorded involtage clamp with a whole cell pipette (holding potential=−60 mV). Thisrecording is typical of 9 of 12 cells examined under these conditions.The frequency of postsynaptic events (PSCs) was greatly increased (up to+400%) by 1 μM hcrt2 applied to the bath. After washout of the peptide,the frequency of PSCs returned to normal baseline levels. Inset boxesshow higher resolution of the events indicated by the dotted line. Bothboxes (in FIG. 7 A and B) were recorded with an identical delay afterhcrt2 administration. A less mature hypothalamic neuron after 4 days ofculture was unresponsive to 1 μM hcrt2 (FIG. 7B). Pipette solutioncontained 128 mM KMeSO₄, 27 mM KCl, 0.4 mM EGTA, 1 mM ATP, and 0.5 mMGTP.

[0343] The preceding written description provides a full, clear, conciseand exact disclosure of the invention so as to enable one skilled in theart to make and use the same. This disclosure should not be construed soas to impart any direct or implied limitation upon the scope of theinvention which is particularly pointed out and distinctly claimedbelow. TABLE 1 Cumulative Data From 100 Clones Clone^(a) BLASTHomology^(b) Accession#^(c) #^(d) Pattern^(e)  2 + oxytocin M25649 13A/A  6 + VAT1-like T05306 11 B/B  1 + CART U10071 7 C 35 + novel 6 A/A15 + novel 4 B/C 25 + POMC J00759 4 A 12 + novel(E) R75926 3 B/B 16 +vasopressin M25646 3 A 18 + glutathione perox U13705 3 B 29 + novel CaMkinase 3 B/C  3 + novel 2 B/C 10 + novel 2 B/B 51 + ubiquitin carrierM91679 2 C 62 + novel 2 C  5 − calbindin U08290 B 14 + melanin-conchormone M62641 C 17 + asp aminotrans M18467 D 19 − novel(E) R74893 D20 + novel A/B 21 − novel(E) T32756 A/D 22 − novel D 33 + novel(E)R67552 A/A 34 − Cl³¹/HCO₃ ⁺exchanger J05167 C 37 + novel B/D 39 − novelC 45 + novel C 46 + fibromodulin X82152 C 47   perox enolhydrataseU08976 C 48 + galanin J03624 B 52 − 5-HT₂ receptor L31546 B 53 + MHC orfM32010 E 55 + HNF dimer cofactor M83740 C 56 + carbonyl reductase X84349C 57 + tyrosine hydroxylase M10244 A 63 + novel D 67 + novel B/B 73 +novel C 74 + novel(E) T93996 C 75 + lamin C2 D14850 A 86 + novel C 92 −novel(E) R49544 C 98 + novel B/D 99 − neuronal kinesin U06698 B/D

[0344]

1 15 130 amino acids amino acid <Unknown> unknown protein NO NO 1 MetAsn Leu Pro Ser Thr Lys Val Pro Trp Ala Ala Val Thr L 1 5 10 15 Leu LeuLeu Leu Leu Pro Pro Ala Leu Leu Ser Leu Gly Val A 20 25 30 Gln Pro LeuPro Asp Cys Cys Arg Gln Lys Thr Cys Ser Cys A 35 40 45 Tyr Glu Leu LeuHis Gly Ala Gly Asn His Ala Ala Gly Ile L 50 55 60 Leu Gly Lys Arg ArgPro Gly Pro Pro Gly Leu Gln Gly Arg L 65 70 75 80 Arg Leu Leu Gln AlaAsn Gly Asn His Ala Ala Gly Ile Leu T 85 90 95 Gly Arg Arg Ala Gly AlaGlu Leu Glu Pro Tyr Pro Cys Pro G 100 105 110 Arg Cys Pro Thr Ala ThrAla Thr Ala Leu Ala Pro Arg Gly G 115 120 125 Arg Val 130 130 aminoacids amino acid <Unknown> unknown protein NO NO 2 Met Asn Phe Pro SerThr Lys Val Pro Trp Ala Ala Val Thr L 1 5 10 15 Leu Leu Leu Leu Leu ProPro Ala Leu Leu Ser Leu Gly Val A 20 25 30 Gln Pro Leu Pro Asp Cys CysArg Gln Lys Thr Cys Ser Cys A 35 40 45 Tyr Glu Leu Leu His Gly Ala GlyAsn His Ala Ala Gly Ile L 50 55 60 Leu Gly Lys Arg Arg Pro Gly Pro ProGly Leu Gln Gly Arg L 65 70 75 80 Arg Leu Leu Gln Ala Asn Gly Asn HisAla Ala Gly Ile Leu T 85 90 95 Gly Arg Arg Ala Gly Ala Glu Leu Glu ProHis Pro Cys Ser G 100 105 110 Gly Cys Pro Thr Val Thr Thr Thr Ala LeuAla Pro Arg Gly G 115 120 125 Gly Val 130 569 base pairs nucleic acidsingle unknown cDNA NO NO 3 TAAGACGACG GCCTCAGACT CCTTGGGTAT TTGGACCACTGCACCGAAGA TACCA60 CCGGATTACC TCTCCCTGAG CTCCAGACAC CATGAACCTTCCTTCTACAA AGGT120 GGCCGCCGTG ACGCTGCTGC TGCTGCTACT GCTGCCGCCGGCGCTGCTGT CGCT180 GGACGCGCAG CCTCTGCCCG ACTGCTGTCG CCAGAAGACGTGTTCCTGCC GGCT240 ACTGTTGCAC GGAGCTGGCA ACCACGCCGC GGGCATCCTCACTCTGGGAA AGCG300 TGGACCCCCA GGCCTCCAAG GACGGCTGCA GCGCCTCCTTCAGGCCAACG GTAA360 AGCTGGCATC CTGACCATGG GCCGCCGCGC AGGCGCAGAGCTAGAGCCAT ATCC420 TGGTCGCCGC TGTCCGACTG CAACCGCCAC CGCTTTAGCGCCCCGGGGCG GATC480 CTGAACCCGT CTTCTATCCC TGTCCTAGTC CTAACTTTCCCCTCTCCTCG CCAG540 GGCAATAAAG ACGTTTCTCT GTTGGTGTG 569 582 base pairsnucleic acid single linear cDNA NO NO N-terminal 4 TAAGACGACG GCCTCAGACTTCTTGGGTAT TTGGACCACT GCACTGAAGA GATCA60 CCAGATTACT TTCCCCTGAGCTCCAGGCAC CATGAACTTT CCTTCTACAA AGGT120 GGCCGCCGTG ACGCTGCTGCTGCTGCTACT GCTGCCACCG GCGCTGCTGT CGCT180 GGACGCACAG CCTCTGCCCGACTGCTGTCG CCAGAAGACG TGTTCCTGCC GTCT240 ACTGTTGCAC GGAGCTGGCAACCACGCTGC GGGTATCCTG ACTCTGGGAA AGCG300 TGGACCTCCA GGCCTCCAGGGACGGCTGCA GCGCCTCCTT CAGGCCAACG GTAA360 AGCTGGCATC CTGACCATGGGCCGCCGCGC AGGCGCAGAG CTAGAGCCAC ATCC420 TGGTCGCGGC TGTCCGACCGTAACTATCAC CGCTTTAGCA CCCCGGGGAG GGTC480 TTGAACCCAT CTTCTATCCTTGTCCTGATC CAAACTTCCC CCTCTGCTCG CCGC540 TCTCTTGGTA AATGGCAATAAAGACGTTTC TCTGTTGGTG TG 582 1458 base pairs nucleic acid single linearcDNA NO NO N-terminal 5 GCTAGGAGAC ATTGCGGCGG CGGTGGCGGC GTTGGCAGCAGCTGCAGACA TGCTG60 CAAGAAACAG ACGGAGGACA TCAGCAGTGT CTATGAGATCCGGGAGAAGC TGGG120 TGCCTTCTCT GAGGTGATGC TGGCCCAGGA AAGGGGCTCTGCTCATCTTG TGGC180 GTGCATTCCC AAGAAAGCAC TTCGGGGCAA GGAGGCCCTGGTGGAGAATG AGAT240 ACTCCGCAGG ATTAGCCACC CCAACATTGT GGCTCTGGAGGACGTCCACG AGAG300 CCATCTCTAC TTGGCCATGG AGCTGGTAAC AGGTGGTGAACTGTTTGACC GAAT360 GCGGGGCTCC TACACAGAGA AGGATGCGAG CCACCTTGTAGGGCAGGTCC TTGG420 CTCCTACCTT CATAGCCTGG GCATCGTGCA CCGGGACCTCAAGCCTGAAA ACCT480 TGCCACACCT TTTGAGGACT CCAAGATCAT GGTCTCTGACTTTGGCCTGT CCAA540 AGCTGGCAAC ATGCTAGGCA CAGCCTGTGG GACCCCAGGATATGTGGCCC CAGA600 GGAGCAGAAA CCCTACGGGA AGGCCGTAGA TGTGTGGGCCCTGGGTGTCA TCTC660 CCTGCTGTGT GGGTACCCCC CCTTCTATGA TGAGAGCGATCCTGAACTCT TCAG720 TCTGAGGGCC AGCTACGAGT TTGACTCTCC CTTTTGGGATGACATCTCAG AATC780 AGACTTCATT CGGCACCTTC TGGAACGTGA TCCCCAGAAGAGGTTCACCT GCCA840 CTTACAGCAT CTCTGGATCT CTGGGGATGC AGCCTTGGACAGGGACATCC TAGG900 CAGTGAGCAG ATCCAGAAGA ATTTTGCCAG GACCCACTGGAAGCGTGCAT TCAA960 ATCATTCCTA CGTCACATCC GTAAGCTGGG ACAGAGCCCAGAGGGTGAGG AGG1020 GCAGGGTATG ACCCGTCACA GCCACCCAGG CCTTGGGACTAGCCAGTCTC CCA1080 ACAACCAGGT GGATGCCAAG GAAGGCCAAG TGGACTGACTCCTAGCTTTT CTT1140 GCCCTTTTGA TCTCCTTCCC TGATCCTTGT CCCCCGGACTGGCCTCTGTT GGA1200 AGACCGTGGG TGTGATGCAT GGCACTGGGG TATGGGGCTTCCCAAGTATG TCC1260 CTGTCCTTTG TTGCTGCCAC CCTCTATGGA AACTGAGGAGGTATTCAAAA ATG1320 GGGCCATCCT TCCTGCACCT TGCACGCACA TATGCATTGCGTGGCTGTTC TGT1380 TGACTGTGGG TGGTCCTGCT TGTGTTGTAG CCCTTTAGTTCCTCCTCTTT CCA1440 AAGACAAACA GAACAATG 1458 103 amino acids amino acid<Unknown> unknown protein NO NO 6 Leu Gly Val Asp Ala Gln Pro Leu ProAsp Cys Cys Arg Gln L 1 5 10 15 Cys Ser Cys Arg Leu Tyr Glu Leu Leu HisGly Ala Gly Asn H 20 25 30 Ala Gly Ile Leu Thr Leu Gly Lys Arg Arg ProGly Pro Pro G 35 40 45 Gln Gly Arg Leu Gln Arg Leu Leu Gln Ala Asn GlyAsn His A 50 55 60 Gly Ile Leu Thr Met Gly Arg Arg Ala Gly Ala Glu LeuGlu P 65 70 75 80 Pro Cys Pro Gly Arg Arg Cys Pro Thr Ala Thr Ala ThrAla L 85 90 95 Pro Arg Gly Gly Ser Arg Val 100 39 amino acids amino acid<Unknown> unknown protein NO NO 7 Leu Gly Val Asp Ala Gln Pro Leu ProAsp Cys Cys Arg Gln L 1 5 10 15 Cys Ser Cys Arg Leu Tyr Glu Leu Leu HisGly Ala Gly Asn H 20 25 30 Ala Gly Ile Leu Thr Leu Gly 35 38 amino acidsamino acid <Unknown> unknown protein NO NO 8 Leu Gly Val Asp Ala Gln ProLeu Pro Asp Cys Cys Arg Gln L 1 5 10 15 Cys Ser Cys Arg Leu Tyr Glu LeuLeu His Gly Ala Gly Asn H 20 25 30 Ala Gly Ile Leu Thr Leu 35 28 aminoacids amino acid <Unknown> unknown protein NO NO 9 Pro Gly Pro Pro GlyLeu Gln Gly Arg Leu Gln Arg Leu Leu G 1 5 10 15 Asn Gly Asn His Ala AlaGly Ile Leu Thr Met Gly 20 25 27 amino acids amino acid <Unknown>unknown protein NO NO 10 Pro Gly Pro Pro Gly Leu Gln Gly Arg Leu Gln ArgLeu Leu G 1 5 10 15 Asn Gly Asn His Ala Ala Gly Ile Leu Thr Met 20 25 20amino acids amino acid <Unknown> unknown protein NO NO 11 Arg Leu TyrGlu Leu Leu His Gly Ala Gly Asn His Ala Ala G 1 5 10 15 Leu Thr Leu Gly20 20 amino acids amino acid <Unknown> unknown protein NO NO 12 Arg LeuGln Arg Leu Leu Gln Ala Asn Gly Asn His Ala Ala G 1 5 10 15 Leu Thr MetGly 20 9 amino acids amino acid <Unknown> unknown protein NO NO 13 GlyAsn His Ala Ala Gly Ile Leu Thr 1 5 393 base pairs nucleic acid singlelinear cDNA NO NO N-terminal 14 ATGAACCTTC CTTCTACAAA GGTTCCCTGGGCCGCCGTGA CGCTGCTGCT GCTGC60 CTGCCGCCGG CGCTGCTGTC GCTTGGGGTGGACGCGCAGC CTCTGCCCGA CTGC120 CAGAAGACGT GTTCCTGCCG TCTCTACGAACTGTTGCACG GAGCTGGCAA CCAC180 GGCATCCTCA CTCTGGGAAA GCGGCGACCTGGACCCCCAG GCCTCCAAGG ACGG240 CGCCTCCTTC AGGCCAACGG TAACCACGCAGCTGGCATCC TGACCATGGG CCGC300 GGCGCAGAGC TAGAGCCATA TCCCTGCCCTGGTCGCCGCT GTCCGACTGC AACC360 GCTTTAGCGC CCCGGGGCGG ATCCAGAGTC TGA 393393 base pairs nucleic acid single linear cDNA NO NO N-terminal 15ATGAACTTTC CTTCTACAAA GGTTCCCTGG GCCGCCGTGA CGCTGCTGCT GCTGC60CTGCCGCCGG CGCTGCTGTC GCTTGGGGTG GACGCACAGC CTCTGCCCGA CTGC120CAGAAGACGT GTTCCTGCCG TCTCTACGAA CTGTTGCACG GAGCTGGCAA CCAC180GGTATCCTGA CTCTGGGAAA GCGGCGGCCT GGACCTCCAG GCCTCCAGGG ACGG240CGCCTCCTTC AGGCCAACGG TAACCACGCA GCTGGCATCC TGACCATGGG CCGC300GGCGCAGAGC TAGAGCCACA TCCCTGCTCT GGTCGCGGCT GTCCGACCGT AACT360GCTTTAGCAC CCCGGGGAGG GTCCGGAGTC TGA 393

We claim:
 1. An isolated polynucleotide selected from the groupconsisting of: (a) a polynucleotide encoding the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1; (b) a polynucleotide encodingthe polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (c)a polynucleotide capable of hybridizing to and which is at least 95%homologous to the polynucleotide of (a) or (b).
 2. The polynucleotide ofclaim 1 comprising the sequence of the group consisting of the secondline of FIG. 5 and SEQ ID NO:
 3. 3. The polynucleotide of claim 1comprising the sequence of the group consisting of the third line ofFIG. 5 and SEQ ID NO:
 4. 4. An isolated polynucleotide selected from thegroup consisting of: (a) a polynucleotide encoding a polypeptidecomprising the sequence of SEQ ID NO: 6; (b) a polynucleotide encoding apolypeptide comprising amino acids 28 to 130 of SEQ ID NO: 2; (c) apolynucleotide capable of hybridizing to the polynucleotide of (a); (d)a polynucleotide capable of hybridizing to the polynucleotide of (b);(e) a polynucleotide that is at least 95% homologous to thepolynucleotide of (a); and (f) a polynucleotide that is at least 95%homologous to the polynucleotide of (b).
 5. An isolated polynucleotideselected from the group consisting of: (a) a polynucleotide encoding apolypeptide comprising the sequence of SEQ ID NO: 7; (b) apolynucleotide encoding a polypeptide comprising the sequence of SEQ IDNO: 8; (c) a polynucleotide encoding a polypeptide comprising aminoacids 42 to 66 of SEQ ID NO: 1; (d) a polynucleotide encoding apolypeptide comprising amino acids 42 to 65 of SEQ ID NO: 1; (e) apolynucleotide encoding a polypeptide comprising amino acids 43 to 66 ofSEQ ID NO: 1; (f) a polynucleotide encoding a polypeptide comprisingamino acids 43 to 65 of SEQ ID NO: 1; and (g) a polynucleotide capableof hybridizing to and which is at least 95% homologous to apolynucleotide of (a) through (f).
 6. An isolated polynucleotideselected from the group consisting of: (a) a polynucleotide encoding apolypeptide comprising the sequence of SEQ ID NO: 9; (b) apolynucleotide encoding a polypeptide comprising the sequence of SEQ IDNO: 10; and (c) a polynucleotide capable of hybridizing to and which isat least 95% homologous to the polynucleotide of (a) or (b).
 7. Anisolated polynucleotide selected from the group consisting of: (a) apolynucleotide encoding a polypeptide comprising amino acids 100 to 130of SEQ ID NO: 1; (b) a polynucleotide encoding a polypeptide comprisingamino acids 100 to 130 of SEQ ID NO: 2; (c) a polynucleotide encoding apolypeptide comprising amino acids 100 to 111 of SEQ ID NO: 1; (d) apolynucleotide encoding a polypeptide comprising amino acids 100 to 110of SEQ ID NO: 1; (e) a polynucleotide encoding a polypeptide comprisingamino acids 114 to 130 of SEQ ID NO: 1; and (f) a polynucleotide capableof hybridizing to and which is at least 95% homologous to apolynucleotide of (a) through (e).
 8. An isolated polypeptide selectedfrom the group consisting of the amino acid sequence of SEQ ID NO: 1 andthe amide thereof.
 9. An isolated polypeptide selected from the groupconsisting of the amino acid sequence of SEQ ID NO: 2 and the amidethereof.
 10. An isolated polypeptide selected from the group consistingof a polypeptide comprising the sequence of SEQ ID NO: 6, a polypeptidecomprising amino acids 28 to 130 of SEQ ID NO: 2, and the amidesthereof.
 11. An isolated polypeptide comprising the sequence of SEQ IDNO:
 13. 12. The isolated peptide of claim 11 wherein the isolatedpeptide is from about 28 amino acid residues to about 39 amino acidresidues long.
 13. An isolated polypeptide selected from the groupconsisting of: (a) a polypeptide comprising the sequence of SEQ ID NO:7; (b) a polypeptide comprising the sequence of SEQ ID NO: 8; (c) apolypeptide comprising amino acids 42 to 66 of SEQ ID NO: 1; (d) apolypeptide comprising amino acids 42 to 65 of SEQ ID NO: 1; (e) apolypeptide comprising amino acids 43 to 66 of SEQ ID NO: 1; (f) apolypeptide comprising amino acids 43 to 65 of SEQ ID NO: 1; (g) apolypeptide comprising at least one conservative amino acid substitutionin the sequence of polypeptides (a-f); and (h) the amides thereof. 14.An isolated polypeptide selected from the group consisting of: (a) apolypeptide comprising the sequence of SEQ ID NO: 9; (b) a polypeptidecomprising the sequence of SEQ ID NO: 10; and (c) the amides thereof.15. An isolated polypeptide selected from the group consisting of: (a) apolypeptide comprising amino acids 100 to 130 of SEQ ID NO: 1; (b) apolypeptide comprising amino acids 100 to 130 of SEQ ID NO: 2; (c) apolypeptide comprising amino acids 100 to 111 of SEQ ID NO: 1; (d) apolypeptide comprising amino acids 100 to 110 of SEQ ID NO: 1; (e) apolypeptide comprising amino acids 114 to 130 of SEQ ID NO: 1; (f) apolypeptide comprising at least one conservative amino acid substitutionin the sequence of polypeptides (a-e); and (g) the amides thereof.
 16. Avector comprising a polynucleotide of claim 1 operably linked to controlsequences which direct the expression of the polynucleotide.
 17. Avector comprising a polyfnucleotide of claim 2 operably linked tocontrol sequences which direct the expression of the polynucleotide. 18.A vector comprising a polynucleotide of claim 3 operably linked tocontrol sequences which direct the expression of the polynucleotide. 19.A vector comprising a polynucleotide of claim 4 operably linked tocontrol sequences which direct the expression of the polynucleotide. 20.A vector comprising a polynucleotide of claim 5 operably linked tocontrol sequences which direct the expression of the polynucleotide. 21.A vector comprising a polynucleotide of claim 6 operably linked tocontrol sequences which direct the expression of the polynucleotide. 22.A vector comprising a polynucleotide of claim 7 operably linked tocontrol sequences which direct the expression of the polynucleotide. 23.A host cell transformed with a vector of claim
 16. 24. A host celltransformed with a vector of claim
 17. 25. A host cell transformed witha vector of claim
 18. 26. A host cell transformed with a vector of claim19.
 27. A host cell transformed with a vector of claim
 20. 28. A hostcell transformed with a vector of claim
 21. 29. A host cell transformedwith a vector of claim
 22. 30. A pharmaceutical composition comprising apolypeptide of claim 8 and a pharmaceutically acceptable carrier.
 31. Apharmaceutical composition comprising a polypeptide of claim 9 and apharmaceutically acceptable carrier.
 32. A pharmaceutical compositioncomprising a polypeptide of claim 10 and a pharmaceutically acceptablecarrier.
 33. A pharmaceutical composition comprising a polypeptide ofclaim 11 and a pharmaceutically acceptable carrier.
 34. A pharmaceuticalcomposition comprising a polypeptide of claim 14 and a pharmaceuticallyacceptable carrier.
 35. A pharmaceutical composition comprising apolypeptide of claim 15 and a pharmaceutically acceptable carrier.
 36. Amethod of treating a neurological disease or homeostatic dysfunction orcontrolling the production of a homeostatic regulatory hormonecomprising introducing an effective amount of the composition of claim18 into a mammal in need of such treatment.
 37. An antibody thatimmunoreacts with an isolated mammalian H35 protein.
 38. An antibody ofclaim 37 which is a monoclonal antibody.
 39. A kit for detecting thepresence of an H35 protein in a mammalian sample comprising an antibodywhich immunoreacts with a mammalian H35 protein or with a polypeptide ofclaim 10 in an amount sufficient for at least one assay and suitablepackaging material.
 40. A kit for detecting the presence of an H35protein in a mammalian sample comprising an antibody which immunoreactswith a mammalian H35 protein or with a polypeptide of claim 11 in anamount sufficient for at least one assay and suitable packagingmaterial.
 41. A kit for detecting the presence of an H35 protein in amammalian sample comprising an antibody which immunoreacts with amammalian H35 protein or with a polypeptide of claim 14 in an amountsufficient for at least one assay and suitable packaging material.
 42. Akit for detecting the presence of an H35 protein in a mammalian samplecomprising an antibody which immunoreacts with a mammalian H35 proteinor with a polypeptide of claim 15 in an amount sufficient for at leastone assay and suitable packaging material.
 43. The kit of claim 39further comprising a detecting antibody which binds to the anti-H35antibody.
 44. The kit of claim 43 wherein the detecting antibody islabeled.
 45. The kit of claim 44 wherein the label comprises enzymes,radioisotopes, fluorescent compounds, colloidal metals, chemiluminescentcompounds, phosphorescent compounds, or bioluminescent compounds.
 46. Akit for detecting the presence of genes encoding an H35 proteincomprising a polynucleotide of claim 1, or fragment thereof having atleast 10 contiguous bases, in an amount sufficient for at least oneassay, and suitable packaging material.
 47. A method for detecting thepresence of a nucleic acid encoding an H35 protein in a mammaliansample, comprising the steps of: (a) hybridizing a polynucleotide ofclaim 1, or fragment thereof having at least 10 contiguous bases, withthe nucleic acid of the sample; and (b) detecting the presence of thehybridization product.
 48. A method of detecting an H35 antigen in amammalian sample comprising the steps of: (a) contacting the sample withan anti-H35 antibody which immunoreacts with a hypocretin polypeptide;and (b) detecting the presence of an immunoreaction complex.
 49. Themethod of claim 48 wherein said complex is detected by admixing saidimmunoreaction complex with a detecting antibody capable ofimmunoreacting with said anti-H35 antibody.
 50. The method of claim 49wherein the detecting antibody is labeled.
 51. The method of claim 48wherein the anti-H35 antibody is immobilized on a solid support.
 52. Themethod of claim 48 wherein the sample comprises cells.
 53. The method ofclaim 52 wherein the cells are peripheral blood mononuclear cells. 54.The method of claim 48 wherein the immunoreaction complex of step (b) isdetected by flow cytometry.
 55. The method of claim 48 wherein theimmunoreaction complex of step (b) is detected by ELISA.
 56. A method ofclaim 48 wherein the immunoreaction complex of step (b) is detected byimmunoblot analysis.
 57. A polynucleotide comprising the sequence of SEQID NO:
 5. 58. A vector comprising the sequence of SEQ ID NO:
 5. 59. Ahost cell transfected with the vector of claim 58.